BRPI0710023A2 - methods and compositions for vaccination of poultry - Google Patents

Abstract

<B> METHODS AND COMPOSITIONS FOR DOMESTIC BIRD VACCINATION <D> The present invention provides methods of inducing an immune response against the Clostridium species in birds, to protect birds from Clostridium infection, and / or to protect birds from related disorders, such as necrotic enteritis. The methods may be practiced on egg and / or after hatching. The invention further provides compositions and methods for delivering an egg composition of this invention directly to the body of the embryo.

Description

Report of the Invention Patent for "ECOMPOSITION METHODS FOR VACCINATION OF DOMESTIC BIRDS". PRIORITY STATEMENT

The present application claims the benefit under U.S.C. § 119 (e) of U.S. Provisional Application No. 60 / 787,567, filed March 30, 2006, the entire contents of which are incorporated by reference herein.

FIELD OF INVENTION

The present invention provides compositions and methods for producing an immune response in avian patients by delivering an egg immunizing composition directly to the avian patient's embryo. The present invention further provides immunogenic compositions and methods for producing an immune response to the Clostridium species in avian patients. protect poultry patients from Clostridium infection, and to protect poultry patients from related disorders, such as necrotic enteritis.

BACKGROUND OF THE INVENTION

Egg vaccination provides several advantages for the poultry industry over current control methods and potential avaccination after hatching, including the potential for uniform automation distribution; co-administration with other egg vaccines, thereby reducing bird handling after hatching, and reduced use of antibiotics.

The discovery that certain vaccines (eg, inactivated or non-replicating vaccines) can be distributed to the embryo's body to produce a strong immune response (similar or better than expected when vaccinating birds on hatching day) allows the development of automation devices. which can target the embryo body (specifically avoiding the amniotic fluid surrounding the embryo body) during the egg application of such vaccines. In addition, knowing that inactivated vaccines should preferably be distributed to the embryo body allows the development of vaccine compositions that are more compatible with injection into the embryo body. Prior to this discovery, it would not have been known or expected that inactivated vaccines would need to be preferably administered to the body of the embryo to produce a strong immune response.

Thus, the present invention is a prior art improvement, providing a more efficient way to administer inactive (killed) vaccines in egg. Prior to this invention, there was no indication that administering the inactive vaccine to embryonic fluids surrounding the embryo's body (amniotic fluid) was anything other than administering the inactivated vaccine to the embryo's body. The present invention demonstrates that inactivated vaccines need to be delivered to the embryo's body properly rather than to the fluids surrounding the embryo's body. Knowing that the embryo's body is an appropriate target for optimal efficacy allows the development of inactivated vaccines and delivery methods for the egg route. The invention includes the administration of immunogenic egg decompositions to poultry and other avian species.

Thus, the present invention fulfills a technical need for improved immunogenic compositions for egg delivery to the embryo and methods for inducing an immune response in birds, for protecting birds from avian and other pathogen infection and / or contamination, and for protecting birds from related disorders.

Clostridium perfringens is associated with several poultry diseases, most notably necrotic enteritis (C. perfringenstype A and type C), but also including cholangioepatitis, cellulitis, gizzard abrasions, and navel infections. Although these bacteria represent a component of normal intestinal flora, several factors may predispose birds to disease development. Such factors include a diet having high levels of fishmeal, wheat, barley or rye; bed straw in high humidity; or exposure to coccidia. Clinically, signs of illness usually include diarrhea, decreased appetite, intestinal damage, depression, and mortality. Traditionally, these diseases are controlled by food antibiotics; however, overuse of antibiotics may result in the development of antibiotic resistant bacterial strains, which pose a significant health risk to humans and animals. Moreover, in certain jurisdictions, the use of antibiotics is highly disadvantaged or even prohibited.

Other methods for controlling Clostridium include a diet that avoids ingredients that irritate the intestinal mucosa, for example a soybean feed; a low moisture bed straw having absorbent material such as wood chips or rice husks; o Uses a competitive exclusion product to maintain a healthy intestinal damicroflora balance, for example Primalac (Star-Labs / Forage Research, Inc., Clarksdale, MO), AVIGUARD® (Bayer Corporation, Kansas City, MO), and BIO- MOS® (Alltech, Inc., Nicholasville, KY); and preventive acidification or severe disinfection of water to minimize losses during a period of illness. Vaccination has been used to control clostridial diseases in other species, including cattle, sheep, goats and porcine. Two main types of C. perfringens vaccines have been developed for non-poultry species: toxoids (inactivated toxins) and bacterinoxoids (inactivated bacterial cultures ("dead") and inactivated toxins). Antitoxins (toxin-specific antibodies) are also used to prevent and treat clostridial diseases in non-domestic birds.

To the inventors be aware, there is no previous report describing the administration in egg (ie, to an egg containing a developing avian embryo) of a C. perfringens vaccine. The use of toxoid or toxoid-bacterin vaccines in eggs is uncertain because such vaccines usually require the use of adjuvants, which may be harmful to the embryo, may inactivate live vaccines against other organisms that are typically administered during the egg period (eg. , vaccinate against Marek's disease), and / or may be incompatible with existing egg injection equipment. Egg vaccination against C. perfringens provides several advantages for the poultry industry over current control methods and avaccination after potential hatching of birds, including the potential for uniform distribution by automation; co-administration with other egg vaccines, thereby reducing bird handling after hatching; and the decreased use of antibiotics.

Thus, there is a need in the art for improved immunogenic compositions and methods for inducing an immune response against Clostridium in birds, to protect birds from Clostridium infection, and to protect birds from related disorders such as necrotic aenteritis.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a method of immunizing an avian patient against a pathogen (e.g., an avian pathogen or a non-avian bird-borne pathogen), comprising administering to the egg during the final incubation room an immunizing dose. of a composition that induces an immune response against the pathogen, where the immunogenic composition is administered by injection into an egg directly into the body of the embryo.

In further embodiments of the invention, the composition induces an immune response to treat and / or prevent infection and / or contamination of the bird resulting from exposure to, or contact with, pathogens causing the following non-limiting examples of diseases, infections and / or disorders: coccidiosis, Marek's disease, bursal infectious disease, Newcastle disease, avian smallpox infection, Clostridium spp. infection, avian influenza, infectious bronchitis, chick daanemia virus infection, avian yaryngotracheitis, avian metapneumovirus infection, reovirus infection , avian adenovirus infections, rotavirus infection, astrovirus infection, enclosing body hepatitis, posture fall syndrome, adenovirus infection, Escherichia coli infection, Mycoplasma spp. infection, Salmonellaspp infection, Campylobacter spp. infection, Listeria spp. infection, Haemophillus infection spp., Pasteurella spp. and any combination of these.

In still further embodiments of the invention, the composition may comprise, consist essentially of, and / or consist of, a non-replicating agent that induces an immune response against an avian pathogen and / or a pathogen that causes foodborne diseases such as Salmonella infection. spp., Campylobacter spp. infection, Listeria spp. infection, Eseheriehia coli infection, etc., as are known in the art.

The present invention further comprises methods wherein an effective immunizing dose of two or more compositions that elicit an immune response against the avian pathogen is administered to the embryo egg, where the two or more compositions are administered simultaneously or sequentially in any order.

Additionally, methods are provided herein where an effective immunizing dose of two or more compositions that elicit an immune response against the pathogen is administered in egg and at least one composition is administered to the embryo, where the two or more compositions are administered simultaneously or sequentially in any order. .

The present invention provides methods of inducing an immune response against Clostridium species (e.g., Clostridiumperfringens) in birds, to protect birds from Clostridium infection, and / or to protect birds from related disorders, such as enteritenecrotic. The methods may be practiced on egg and / or after hatching. The present invention further provides immunogenic compositions to induce an immune response against Clostridium species in birds, to protect birds from Clostritium infection, and / or to protect birds from related disorders, such as necrotic enteritis.

Thus, as an aspect, the present invention provides a method of immunizing an avian patient (e.g., a small picture) against necrotic enteritis, comprising administering to an egg (for example, during the final quarter of incubation) an effective immunizing dose which provides induces an immune response against Clostridium perfringens, where the immunogenic composition is administered by egg injection. In certain embodiments, the immunogenic composition is administered to the amnion or embryo. The method may optionally be practiced in combination with other immunization regimens (e.g. vaccination against infectious bursal disease, Marek's disease, Newcastle disease and / or coccidiosis) and / or egg feeding of a nutrient and / or modulating formulation. enteric.

As another aspect, the invention provides an immunogenic composition comprising an effective immunizing dose of an attenuated Clostridium species in a pharmaceutically acceptable carrier. In certain embodiments, the immunogenic composition further comprises an adjuvant, which may be, for example, a deposition adjuvant. Representative adjuvants of this invention include, but are not limited to, an aluminum salt, such as aluminum hydroxide gel (alum), aluminum phosphate, or algamulin, and / or may also be a calcium, magnesium, iron and mineral salt or gels. / or zinc, and / or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationic or anionically derived polysaccharides, or polyphosphazenes, and / or saponins, such as Quil-A, and / or emulsions of oils, such as water in oil and water in oil in water and / or Freund completed or incomplete or any combination thereof. In representative embodiments, the immunogenic composition comprises a water in oil in water emulsion. Optionally, the immunogenic composition may additionally comprise one or more additional agents that induce an immune response against other avian pathogens (e.g., an immune response inducing against Eimeria, infectious disease virus, Marek's disease virus and / or Newcastle's disease virus). ) and / or a nutrient formulation and / or an enteric modulator. One or more additional agents may be immunizing agents that produce a protective immune response against Eimería, infectious bursal disease virus, Marek's disease virus and / or Newcastle's disease virus.

As a further aspect, the invention provides an immunogenic composition comprising in a pharmaceutically acceptable carrier:

(a) an effective immunizing dose of a Clostridium toxoid, a Clostridium bacterin, a Clostridium toxin, or any combination of the foregoing; and

(b) an effective immunizing dose of a decoccidiosis vaccine, a Marek's disease vaccine, a bursal infectious disease vaccine, a Newcastle disease vaccine, a smallpox vaccine, or any combination of the foregoing.

The invention also provides an immunogenic composition comprising in a pharmaceutically acceptable carrier:

(a) an effective immunizing dose of a Clostridium toxoid, a Clostridium bacterin, a Clostridium toxin, or any combination of the foregoing; and

(b) an oil emulsion.

As yet another aspect, the invention provides an immunogenic composition comprising in a pharmaceutically acceptable carrier:

(a) an effective immunizing dose of a Clostridium toxoid, a Clostridium bacterin, a Clostridium toxin, or any combination of the foregoing; and

(b) an adjuvant comprising an aluminum-derived adjuvant, a saponin, an oil, or any combination of the foregoing.

In additional embodiments, the present invention provides a method of immunizing an avian patient against infection with a Clostridium species, comprising administering to the avian patient an effective dose of Clostridium bacterin toxoid composition by injection by egg during the final fourth incubation. In some embodiments of the invention, the Clostridium species may be Clostridiumperfringens.

The present invention also provides a method of immunizing an avian patient against infection with a Clostridium species comprising administering to the avian patient an immunizing-effective dose of a recombinant toxin or its immunogenic fragment of a Clostridium species by injection into an egg during the final fourth incubation. . In some embodiments, the toxin or its immunogenic fragment is a Clostridium perfringens toxin or its immunogenic fragment.

These and other aspects of the invention are set forth in more detail in the description of the invention below.

DETAILED DESCRIPTION OF THE INVENTION

Certain aspects of the present invention are based on the unexpected discovery that certain antigenic or immunogenic compositions allow for a more effective immune response in birds when the composition is administered in an egg directly to the body of the embryo.

Thus, in one embodiment, the present invention provides a method of immunizing an avian patient against a pathogen, which may be an avian pathogen and / or a pathogen that is not avian carried by an avian patient (e.g., a pathogen contained in human food), comprising administering in egg during the incubation quarterfinal an effective immunizing dose of a composition which induces an immune response against the avian pathogen, wherein the immunogenic composition is administered by egg injection directly into the body of the embryo. In the methods of this invention, the composition may be administered directly to the skeletal muscle tissue in the embryo and the skeletal muscle tissue may be, but is not limited to, the chest muscle tissue and mature egg muscle tissue. In additional embodiments, the composition may be administered directly to the embryo on the head, neck, shoulder, wing, back, chest, leg or any combination thereof. Further, in the methods of this invention, the composition may be administered subcutaneously into the body of the embryo. In additional embodiments, the composition may be administered subcutaneously to the head, neck, shoulder, wing, back, chest, leg or any combination thereof.

Any suitable route of administration to the embryo is appropriate in employing the methods of this invention. For example, the composition may be administered to the embryo subcutaneously, intradermally, intravenously, intramuscularly, intra-abdominally or any combination thereof.

The avian patient of this invention may be any bird and, in certain embodiments, the patient may be a chicken, turkey, duck, goose, pheasant, quail, partridge, guinea fowl, ostrich, rhea or peacock, as well as any other commercially available bird. and / or any bird whose eggs are accessible for handling in the methods of this invention.

In embodiments where the patient is a chicken, it is desirable to administer the composition of this invention in egg for the period from day 15 to day 20 of incubation, and in particular embodiments, the composition may be administered on day 18 or day 19 of incubation. If the patient is a turkey, the composition of this invention may be administered from day 21 to day 28 of incubation and, in particular embodiments, the compositions may be administered on day 24 or day 25 of incubation. In other embodiments, where the goose patient, the composition of this invention may be administered during the period from day 23 to day 31 of incubation and, in particular embodiments, the compositions may be administered on day 28 or day 29 of incubation. In additional embodiments, where the patient is a duck, the composition of this invention may be administered during the period from day 21 to day 28 of incubation and, in particular embodiments, the compositions may be administered on day 25 or day 26 of incubation.

For the other avian species, the final fourth incubation, and thus the optimal range of days for egg administration of a composition of this invention, can be determined according to methods well known in the art. For example, a mallard duck has an incubation period in the range of 33-35 days, a common pheasant has an incubation period in the range of 23-24 days, a Japanese quail has an incubation period of 17-18 days, a North American quail has an incubation period of 23 days, a partridge has an incubation period of 22-23 days, a guinea fowl has an incubation period of 26-28 days and a peacock has an incubation period of 28 days

In particular embodiments of this invention, the composition may comprise, consist essentially of, and / or consist of, an immunogenic composition and an adjuvant. The non-limiting examples of this invention include an aluminum-derived adjuvant, unsaponin, mineral gels, polyanions, pluronic polyols, saponin derivatives, lysolecithin and other similar surfactants, glycosides, all types of oils and any combination thereof. In particular embodiments of this invention, the composition may comprise a water-in-water emulsion.

As contemplated herein, in some embodiments of the present invention, the composition of this invention may comprise an adjuvant, which in particular embodiments may be adjuvant such as an aluminum-derived adjuvant (e.g. aluminum hydroxide), a saponin (e.g. Quil-A, including QuiIAQS21), or an oil (such as complete or incomplete Freund's Adjuvant) in any combination.

Additional non-limiting examples of an inventive adjuvant include mineral salts (e.g. aluminum hydroxide; aluminum phosphate; calcium phosphate), oil emulsions and surfactant-based formulations (eg Freund's emulsified oil adjuvants). ; Arlacel A; mineral oil; emulsified peanut oil adjuvant (adjuvant 65); o MF59 (oil emulsion in stabilized microfluidized detergent water); QS21 (purified saponin); oAS02 [SBAS2] (oil in water + MPL + Montanide ISA-51; ISA-720 (stabilized water-in-oil emulsion), bacterial products and derivatives (eg Bordatella pertussis; P40 component derived from Cornebacterlum granulosum; lipopolysaccharide (adjuvant for both humoral and immune-mediated immunity). Mycobacterium and its components (MDPs, adjuvants not acceptable in humans); Cholera toxin (mucosal adjuvant); microbial derivatives (natural and synthetic). ethical) [e.g. monophosphoryl lipid A (MPL); Detox (MPL + M. phlei cell wall skeleton); AGP [RC-529] (synthetic monosaccharide acylate); DC-Chol (lipoidal immunostimulators capable of self-organizing into liposomes); OM-174 (lipid derivative A); CpG motifs (synthetic oligonucleotides containing CpGimostimulatory motifs); Modified LT and CT (genetically engineered bacterial toxins to provide non-toxic adjuvant effects)], endogenous chicken immunomodulators [cytokines; antibodies: hGM-CSF or hlL-12 (cytokines that may be administered as protein or encoded plasmid); Imudaptine (C3d serial arrangement); Squalene], particulate adjuvants [virosomes (unilamellar liposomal carriers incorporating antigen); AS04 ([SBAS4] Al salt with MPL); ISCOMs (structured complex of saponins and lipids); co-glycolide polylactide (PLG) 1 and inert carriers (gold particles; silver particles).

Additional adjuvants of this invention may include mineral gels, polyanions, pluronic polyols, saponin derivatives, alisolecithin and other similar surfactants. Additional adjuvants may include toll-like receptor (TLR) agonists, including, for example, TLR-1 agonists (e.g., tri-acyl lipopeptides); TLR-2 antagonists [e.g., peptidoglycan from gram-positive bacteria such as streptococci and staphylococci; lipoteichoic acid]; TLR-3 antagonists (e.g., double stranded RNA and analogs thereof, such as poly 1: C); TLR-4 agonists [e.g. lipopolysaccharide (endotoxin) from gram negative bacteria such as Salmonella and E. coli]; TLR-5 antagonists (e.g. flagellin from bacteria that have mobility such as Listeria); TLR-6 agonists [e.g., with TLR-2 peptidoglycan and certain lipids (diacyl lipopeptides)]; TLR-7 agonists [e.g., single stranded RNA (ssRNA) genomes of such viruses as influenza, measles, and mumps; and small synthetic guanosine-based antiviral molecules such as Ioxoribine and ssRNA and their analogs]; TLR-8 agonists (e.g., binds ssRNA); TLR-9 agonists (eg, unmethylated CpG from the pathogen's DNA and its analogs; TLR-10 (undefined function) and TLR-11 agonists (eg binds proteins expressed by various infectious protozoans (Apicomplexa). Chickens have a well developed TLR system, with approximately 10 TLRs broadly similar to those detected in mammals.

Further examples of the adjuvants of this invention include complement receptors (secreted PRRs), where C3d (complement component) is activated by microbial CHOs. The complementorus pathway results in pathogen opsonization and rapid phagocytosis.

In additional embodiments, an adjuvant of this invention may be an amino acid sequence that is a peptide, a protein fragment or an entire protein that functions as the adjuvant, or the adjuvant may be a nucleic acid encoding a peptide, protein fragment or whole protein that functions as an adjuvant. an adjuvant. As used herein, the "adjuvant" describes a substance, which may be any immunomodulatory substance capable of being combined with the polypeptide or nucleic acid vaccine to augment, otherwise improved modulating an immune response in a patient, with no effect on the patient.

An adjuvant of this invention may be, but is not limited to, for example, an immunostimulatory cytokine (including, but not limited to, GM / CSF, interleukin-2, interleukin-12, interferon-gamma, interferon-4, tumor necrosis factor alpha, interleukin-1, fit3L hematopoietic factor, CD40L, B7.1 costimulatory molecules and B7.2 costimulatory molecules, SYNTEX 1 (SAF-1) adjuvant formulation composed of 5 percent (weight / volume) squalene ( DASF, Parsippany, NJ), 2.5 percent Pluronic® L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline solution. Suitable adjuvants also include an aluminum salt, such as aluminum hydroxide gel (alum), aluminum phosphate, or algamulin, but may also be a calcium, iron or zinc salt, or may be an insoluble acylated tyrosine suspension, or acylated sugars, cationically or anionically derived polysaccharides, or polyphosphates.

Other adjuvants are well known in the art and include N-acetyl muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor -MDP), α-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine (CGP 19835A, referred to as MTP-PE ) and RIBI, which contains three bacterially extracted components, monophosphorylipid A, trehalose dimicolate and cell wall skeleton (MPL + TDM + CWS) in 2% squalene / Tween 80 emulsion.

Additional adjuvants may include, for example, a combination of monophosphoryl lipid A, preferably A3-des-O-acylated monophosphoryl lipid (3-D-MPL), together with an aluminum salt. An improved adjuvant system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL, as disclosed in PCT publication number WO 94/00153 (the entire contents of which are incorporated herein by reference), or a composition. reactor, where QS21 is terminated with cholesterol, as disclosed in PCT publication number WO 96/33739 (the entire contents of which are incorporated herein by reference). A particularly potent adjuvant formulation involving QS21 3D-MPL and tocopherol in an emulsion of oil in water is described in PCT publication number WO 95/17210 (whose entire contents are incorporated herein by reference).

The compositions and methods of the present invention may be employed to induce an immune response, to treat and / or prevent such diseases and disorders as coccidiosis, Marek's disease, infectious bursal disease, Newcastle disease, avian smallpox infection, Clostridium spp. (eg necrotic enteritis, dermatitegangrenosa, cholangioepatitis, cellulitis, ulcerative enteritis, botulism, Tyzzer disease), avian influenza, infectious bronchitis, chick anemia virus infection, avian laryngotracheitis, avian metapneumovirus, avian reovirus infection, avian adenovirus infections, rotavirus infection, astrovirus infection, body-enclosing hepatitis, posture syndrome, adenovirus infection, Escherichia coii infection, infection Mycoplasma spp., Salmoriella spp. infection, Campylobacter spp. infection, Listeria spp., Haemophillus spp. infection, Pasteureila spp. infection, Staphylococcus spp., Streptoeoeeus spp., Myeobaeterium spp. Erysipelothrix spp. and any combination of these.

Thus, in certain embodiments, the composition of this invention may comprise, consist essentially of, and / or consist of, an antigen or immunogen from Marek's disease virus, infectious dabronchitis virus, Myeoplasma spp., Avian leukosis virus, reovirus, poxvirus. , adenovirus, cryptosporidium, infectious chicken anemia virus, Pasteurella species, avian influenza virus, Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Rous1 dosarcoma virus Eseheriehia eoli, Eimeria species, such as Eimeriatenella (which causes coccidiosis), Haemophilus species, Mycoplasma, Listeria species, Salmonella species, Clostridium Campylobaeteriespecies species (eg C. perfringens, C. septieum, C. sordellii, C. diffieile, C. novyi, C. botulinum, C. eolinum, C. ehauvoei, C. fallax, C.sporogenes, and C. piliform) and any combination thereof.

The present invention is intended to include methods and compositions for immunizing birds against pathogens which may be disease causing pathogens in birds and / or pathogens that are carried by birds (contaminated birds) and are passed on to humans and other animals handling or eat such avescontaminated. Thus, the present invention provides compositions comprising, consisting essentially of, and / or consisting of, a non-replicating agent which induces an immune response against an avian pathogen or a pathogen that causes disease in other animals by contact with or ingestion of eggs or meat or other body parts of, a contaminated bird. Such pathogens may include, but are not limited to, Salmonella spp., Campylobacter spp., Listeria, Escherichia coli, Erysipelothrix sp., Mycobacterium spp., Clostridium spp.

In some embodiments, the methods of the invention described herein may further comprise the step of administering a booster dose of the composition of this invention to the patient after hatching.

In additional embodiments of the methods of this invention, an effective immunizing dose of two or more compositions that induce an immune response against the avian pathogen is administered to the embryo in egg, where the two or more compositions are administered simultaneously or sequentially in any order. Thus, the compositions and methods of this invention may be employed using apparatus and technology which comprises administering multiple compositions in a single location, multiple compositions in multiple locations and / or a single composition in multiple locations. Such methods may employ a single egg entry site or multiple egg entry sites. Non-limiting examples of such apparatus and technology are described in US Patent No. 4,903,635, US Patent No. 5,136,979, US Patent No. RE 35,973, US Patent No. 5,339,766, US Patent No. 6,032,612, US Patent No. 6,286,455, US Patent No. 5,158,038, US Patent No. 6,601,534 and U.S. Patent. No. 6,981,470, the entire contents of which are incorporated by reference in this document.

Also included herein are methods wherein an effective immunizing dose of two or more compositions that elicit an immune response against the avian pathogen is administered to an egg, and at least one composition is administered to the embryo, where the two or more compositions are administered simultaneously or sequentially to any one. order. In some embodiments of these methods, at least one composition may be administered to amnion, which includes the amniotic fluid, embryo body and yolk sac, and / or at least one composition may be administered directly into the amniotic fluid, embryo body and / or in the yolk sac, individually or in any combination.

In some embodiments of this invention, the methods may additionally comprise administering to an egg an immune stimulant at any time during incubation, where the immune stimulant and composition are administered simultaneously or sequentially in any order. The methods of this invention may further comprise administering to egg an nutrient formulation, an enteral modulator, or a combination thereof at any time during incubation, wherein the nutrient formulation, the enteric modulator or a combination thereof and the composition are administered simultaneously or sequentially in any order. .

There are several aspects of avian embryonic development that make the embryo an attractive target for immunization. First, since the longest period of embryonic development occurs in the egg outside the maternal reproductive tract, the embryo can be easily accessed for introducing compositions such as immunogenic compositions. of this invention.

Second, the fact that the egg is a compartmentalized multi-unit can be explored to distribute biological materials to specific embryonic sites. For example, the early embryonic vitelinono sac works to make blood. Immediately prior to hatching, the yolk sac has a mainly nutritional function and is partly carried into the intestinal tract and thereby transported to the cecal sacs during and after hatching. Therefore, administration of materials to the yolk sac may result in distribution to either embryonic cecal or vascular systems. In addition, administration of a composition of this invention can be efficiently effected by injection into the chorioallantoic membrane or the air cell membrane. Finally, access to the embryonic muscle compartment can be achieved by direct embryonic injection in the last fourth incubation transfer, and in the chickens thereof. It is usually performed from day 17 until day 19 of incubation.

The immunogenic composition may be introduced into any region of the egg, including the air cell, albumen, chorioallantoic membrane, yolk sac, yolk, allantoid, amnion, or directly the embryonic bird. In a particular embodiment of the invention, the composition is introduced into the muscle tissue of the embryonic bird, and in another embodiment, the composition is introduced into the skeletal muscle tissue. In certain embodiments, the introduction of a nucleic acid molecule encoding a protein that remains within the small cell can be used to deliver a specific and direct immunogenic protein to muscle cells. Alternatively, a nucleic acid molecule may be introduced which encodes a protein that will be secreted from the muscle cell, and this method may be used to distribute a protein to the entire body of the bird through contact between muscle tissue and plasma. Illustrative sites of introduction into skeletal muscle tissue are the tissues of the chest muscle and mature dovulus muscle, which are located near the eggshell and thus are relatively easily reached by the injection apparatus without damage to other embryonic structures.

Any suitable means for introducing the composition of this invention into egg may be used, including egg injection, high pressure spraying through the eggshell, and the egg bombardment with microparticles carrying the composition. In some embodiments, the composition is administered by depositing a pharmaceutically acceptable aqueous solution into the muscle, which solution contains the composition to be deposited.

Where egg injection is used, the injection mechanism is not critical, but it is preferred that the method does not unduly damage the embryo organs or surrounding embryonic membranes, so treatment will not decrease the rate of injection. outbreak. Preferred injection sites are intramuscular and subcutaneous. Preferred injection sites in the muscle tissue are the skeletal muscle tissues, and more particularly the breast muscle and mature dovulus muscle, which are located near the eggshell and thus are relatively easily reached by the injection apparatus without damage to other embryonic structures and without compromising the protection provided by the eggshell. A syringe fitted with an approximately 18 to 26 scale needle is suitable for the purpose. Depending on the exact stage of development and position of the embryo, a 1.905 to 10.16 centimeter (% to 4 inch) needle will terminate in the fluid above the dopint or chick itself. A pilot hole may be drilled or drilled through the shell prior to needle insertion to prevent needle damage or blindness. If desired, the egg may be sealed with a substantially bacteria-impermeable sealing material, such as wax or the like, to prevent subsequent entry of undesirable bacteria.

In various embodiments of this invention, the composition is administered to the body of the embryo in a needle having a length of about 1.905 centimeters to about 10.16 centimeters (3A inch to 4 inches) [e.g. 2.54 centimeters (1 inch), 3.175 centimeters (1.25 inch), 3.81 centimeters (1.5 inch), 4.445 centimeters (1.75 inch), 5.08 centimeters (2.0 inches), 5.715 centimeters (2.25 inches) ), 6.35 centimeters (2.5 inches), 6.855 centimeters (2.75 inches), 7.62 centimeters (3.0 inches), 8.255 centimeters (3.25 inches), 8.89 centimeters (3.5 inches) inches), 9.525 centimeters (3.75 inches), or 10.16 centimeters (4.0 inches)]. In addition, a needle of this invention may have a scale ranging from 15g to 28g (for example, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g or 28g). In some embodiments, the needle may have a non-pointed end, and in some embodiments, the needle may have a slanted end, with an angle of inclination of about 10 ° to about 45 ° (e.g., 11 °, 12 °, 13 ° , 14 °, 15 °, 16 °, 17 °, 18 °, 19 °, 20 °, 2122 °, 23 °, 24 °, 25 °, 26 °, 27 °, 28 °, 29 °, 30 °, 31 °, 32 °, 33 °, 34 °, 35 °, 36 °, 37 °, 38 °, 39 °, 40 °, 41 °, 42 °, 43 °, 44 °, or 45 °).

In particular embodiments of this invention, in methods of administering an egg composition of this invention, the needle may pass through the shell at the large end of an egg, at an angle from about 1 ° to about 20 ° (e.g., 1 °, 2 °). °, 3 °, 4 °, 5 °, 6 °, 7 °, 8 °, 9 °, 10 °, 11 °, 12 °, 13 °, 14 °, 15 °, 16 °, 17 °, 18 °, 19 °, or 20 °) from the long axis of the new.

The present invention also provides methods wherein the composition of this invention is administered to an egg with an automation injection device.

It is envisaged that a high speed automation injection system for avian embryos will be particularly suitable for practicing the present invention. Several such devices are available, for example the EMBREX INOVOJECT® system (described in U.S. Pat. No. 24,681,063 to Hebrank), and in U.S. Pats. No. 4,040,388, 4,469,047, and 4,593,646 to Miller. The disclosure of these references and all references cited herein is to be incorporated herein by reference. All such devices, as adapted to practice the present invention, comprise a DNA-containing injector, as described herein, with the injector positioned to inject an egg loaded by the apparatus with the DNA. In addition, a sealing apparatus operatively associated with the injection apparatus may be provided to seal the hole in the egg following its injection.

The presently preferred apparatus for practicing the present invention is disclosed in U.S. Pat. No. 4,681,063 to Hebrank and U.S. Pat. No. 4,903,625 to Hebrank, the disclosures of which are incorporated herein by reference. This device comprises an injection device for dispensing fluid substances to a plurality of eggs and a suction apparatus which fits in and simultaneously suspends a plurality of individual eggs from their upwardly facing parts and cooperates with the injector to Inject the eggs while the eggs are fitted into the suction apparatus. The features of this apparatus may be combined with the features of the apparatus described above to practice the present invention. Those skilled in the art will appreciate that this device can be adapted for injection into any part of the egg by adjusting the penetration depth of the injector as discussed in more detail below.

Embodiments of an injection method and apparatus which may be employed in the present invention are described in US Patent No. 26,032,612 (multi-site egg injection apparatus), USN-6,244,214 (Egg injection apparatus and detection of information with respect to the interior of the egg), U.S. Patent Nos. 6,176,199, 6,510,811, and 6,834,615 (Methods of Locating Allantoic Fluid in an Egg), U.S. Pat. No. 7,089,879 (methods for manipulating air cells within new aviaries) and US Patent No. 7,165,507 (methods and apparatus for correctly positioning a device within the subgeminal cavity of an egg), the entire contents of which are incorporated by reference. in this document.

Thus, in some embodiments, the method and apparatus are essentially as described in one or more of the patents listed above, and involve positioning an injector or elongated injection needle at the large end of the egg at an angle deviation (A) from the axis of the said egg, the angle selected so that the needle is directed towards the shoulder or chest of said embryo. The needle is then inserted through the eggshell along an essentially linear displacement path to a depth sufficient to pass into the embryo's shoulder or chest. The substance to be deposited in the egg, which may be a syringe-injectable liquid or solid (e.g., injectable) (but is generally an aqueous solution containing the composition of this invention as described herein), is then injected through the needle. . In some embodiments, the needle is withdrawn along the essentially linear displacement path, and the substance injecting step is performed concurrently with the needle withdrawing step, so that the substance is administered along the displacement path within the egg. The angle of deviation (A) is sufficient to increase the likelihood of injecting into the shoulder or chest muscle.

Typically, the angle is 1 to 20 degrees, and preferably the angle is 2 to 3 degrees. As an example, the needle may be inserted deep enough under the eggshell to pass into or through and through the embryo's shoulder or chest. The apparatus may be modified to include means operably associated with the apparatus for positioning the egg at an angle to the needle to achieve said angle (A), such as by assembling and positioning the needle (s) at an angle to the angle. suction device.

In a particular example, the methods of the present invention may be practiced with the apparatus described in US Patent No. 6,244,214 to Hebrank (whose entire contents are incorporated by reference herein), wherein an apparatus (e.g., a "smart probe") is described. ) to identify the structure and or specific compartment within the egg that is in contact with a needle that has penetrated the egg cascade, and methods for employing the apparatus for distributing the compositions to specific structures and / or compartments within an egg.

Thus, in certain embodiments, the present invention provides a method of introducing a substance into a chicken's egg muscle, comprising: a) obtaining a chicken egg, wherein said egg contains a chicken embryo in its last incubation room, prior to hatch; b) positioning an elongate injection needle at the large end of the egg at an angle deviation about 1 to 5 degrees from the long axis of said egg, the selected angle so that the needle is directed toward the shoulder or breast of said embryo. ; c) inserting said needle through said egg cascade along an essentially linear displacement path to a depth of about 2.2225 centimeters to 3.81 centimeters (7/8 inch to 1.5 inch) in the shoulder or breast of said embryo; and d) injecting said substance into the egg through said needle. In other embodiments, there is provided a method for introducing a substance into the muscle of a young chicken, comprising: a) obtaining a chicken egg, where said egg contains a 17-19 day old chicken embryo; b) positioning an elongated injection needle at the large end of the egg at an angle deviation about 1 to 5 degrees from the long axis of said egg, said angle selected such that the needle is directed towards the shoulder or chest of said embryo; c) inserting said needle through the shell of said egg along an essentially linear displacement path to a depth of about 2.2225 centimeters to 3.81 centimeters (7/8 inch to 1.5 inch) in the middle or breast of said embryo; and d) injecting said substance into the egg through said needle. In such methods, the needle may be inserted deep enough to pass into and through the shoulder or chest of said embryo.

Also provided herein is an apparatus for simultaneously injecting the muscle tissue of chicken embryos into a plurality of eggs during days 17 to 19 of incubation, said device comprising: engaging means for engaging said plurality of eggs; injection means cooperating with said engaging means for inserting an elongated needle through the shells of said eggs along an essentially linear displacement path to a depth of about 2.2225 centimeters to 3.81 centimeters (7/8 inches). inch to 1.5 inch) on the shoulder or chest of said embryo; and positioning means for positioning said elongate injection needle at the large end of said egg, at an angle of about 1 to 5 degrees of deviation from said axle of said egg, so that said needle is directed towards the shoulder or chest of said embryo. In such an apparatus, the engaging means may comprise suction means for simultaneously suspending a plurality of individual eggs.

In further embodiments of this invention, there are provided compositions and methods for inducing an immune response to the Clostridium species in an avian patient. As an example, acute enterotoxemia, called necrotic enteritis, in birds is caused by Clostridiumperfringens (AeC types have been associated with avian disease). Necrotic enteritis can occur when Clostridia-contaminated food and litter are eaten by birds, and the organism grows in the intestine and then spores. This sporulation process causes release of alpha and beta toxins, which are responsible for intestinal necrosis (particularly in the jejunum and ileum). Clinical signs include depression, decreased appetite and diarrhea. Acute mortality may occur. Predisposing factors for C. perfringens infection and enteritenecrotic include diet and damage to the intestinal mucosa. In the context of commercial domesticated birds, most cases of necrotic enteritis occur in two to five week olds. Thus, the particular embodiments of the present invention are directed to the immunogenic compositions and methods that protect birds against necrotic Clostridium perfringense enteritis, for example by reducing infection rates and / or reducing the severity of infection and / or disease.

The present invention will now be described with reference to the particular embodiments of the invention. This invention may, however, be included in different forms and should not be construed as limited to the embodiments set forth herein. Preferably, these embodiments are provided such that this disclosure is efficient and complete, and fully conveys the scope of the invention to those of skill in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in describing the invention in this document is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The terminology used in the description of the invention contained herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "one", "one", "a" and "o" are intended to include plural forms as well, unless the context clearly indicates otherwise.

As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated items listed, as well as the absence of combinations when interpreted in the alternative ("or").

In addition, the term "about" as used herein when referring to a measurable value, such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is intended to include variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0,5%, or even ± 0,1% of the specified quantity.

The terms "avian" and "avian patients" or "avian" and "mild patient" as used herein are intended to include males and females of any avian or bird species, and in particular are intended to include commercially poultry. raised for young animals, meat or as domestic animals. Thus, the terms "avian" and "avian patient" or "avian" or "bird" and "avian patient" include chickens, turkeys, ducks, geese, quail, pheasant, parakeets, parrots, ascacatuas, cockatiels, ostriches, rheas and the like. In particular modalities, the patient is a chicken or a turkey. The commercial model includes chickens and egg-laying poultry, which are bred for meat and egg production respectively.

In particular embodiments, the patient is one in danger of, or susceptible to, infection or disease caused by the species of Clostridium (eg, necrotic enteritis caused by infection with Clostridium perfringens). Risk factors for enteritenecrotic are known in the art and include, but are not limited to dietary factors (eg, a high wheat, barley, rye or fishmeal diet), poor bed straw conditions, and / or exposure to Eimeria (eg natural exposure to live Eimeria flats). Thus, in particular embodiments, inventive compositions and methods may be advantageously employed to reduce the severity and / or incidence of necrotic enteritis among birds that have been vaccinated against coccidiosis or in flocks that are experiencing a coccidiosis epidemic.

Other diseases and disorders caused by bird infection with Clostridium include, but are not limited to, necrotic enteritis, gangrenous dermatitis, cholangioepatitis, cellulitis, ulcerative enteritis, botulism, and Tyzzer disease. Thus, the present invention provides immunogenic compositions and methods of their use to protect birds from infection by, for example, Clostridium perfringens, C. septicum, C. sordellii, C. difficile, C. novyi, C. botulinum, C. colinum , C.chauvoei, C. fallax, C. sporogenes and / or C. piliform.

The avian patient of this invention may be an embryonic live, egg-dwelling bird, or it may be a hatched bird, including newly hatched birds (ie, approximately the first one, two or three days after hatching), teenagers, and adults.

In particular embodiments, the bird is approximately six, five, four, three, two or one week old or less. In other representative embodiments, the avian patient is a never antestrata patient, i.e. had not previously been exposed to the antigen against which immunity is desired.

The term "egg administration" or "egg administration" as used herein, unless otherwise indicated, means administering an immunogenic composition (for example umavacin) to an avian egg containing a living embryo which develops by any means of penetrating the eggshell and introducing the immunogenic composition. Such means of administration include, but are not limited to, egg injection of the immunogenic composition.

The present invention provides methods of administering an immunogenic composition to a patient to induce an immune response against Clostridium species, optionally an immune protective response, in the patient. The immunogenic composition may be administered in any suitable compartment of the egg (e.g., allantoid, sacovitelline, amnion, air cell and / or avian embryo itself), as would be apparent to one skilled in the art. Methods of embryo administration include, without limitation, parenteral administration, such as, for example, subcutaneous, intramuscular, intra-abdominal, intravenous, and / or intraarticular administration. In particular embodiments, the immunogenic composition is administered to the ammonium (e.g., by axially injecting through the large end of the egg).

The immunogenic composition may be administered to the egg by any suitable method. In particular embodiments, the immunogenic composition is administered via injection. The mechanism of injection into the ovum is not critical, but should generally be selected so that the method does not damage the embryo's tissues and organs or the surrounding extra-embryonic membranes in such a way that treatment unduly decreases the hatching rate. A syringe fitted with an approximately 18 to 23 scale needle is generally suitable for the purpose. Examples of needles suitable for this invention include needles having the following scales: scale 18, 19, 20, 21, 22, or 23. A needle of this invention may be at least 1.27 inches (2.12 inches), 2.12 inches ( 5/6 inch), 1.905 centimeter (3rd inch), 2.2225 cm (7/8 inch), 2.54 cm (1 inch), 3.175 cm (1 inch e!), 3.4925 cm (1 inch) inch and 3/8), 3.81 centimeters (1 inch and Vz), 4.1275 centimeters (1 inch and 5/8), 4.445 centimeters (1 inch and%), 4.7625 centimeters (1 inch and 7/8) or 5.08 centimeters (2.0 inches) in length. Examples of a needle bias range of this invention are about 5 degrees to about 45 degrees (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22.23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 45, 56, 37, 38, 39, 40, 41, 42 , 43, 44, or 45 degrees). In certain embodiments of this invention, the inclination range of a needle of this invention is from about 12 degrees to 20 degrees. A pilot hole may be drilled or drilled through the shell prior to insertion of the needle to prevent or damage the needle. If desired, the egg may be sealed with a sealing material substantially impermeable to bacteria such as wax or similar, to prevent subsequent entry of unwanted bacteria.

A high speed automation egg injection system for avian embryos is particularly suitable for practicing the present invention. Several such devices are available, for example those disclosed in U.S. Patent Nos. 4,681,063 and 4,903,635 to Hebrank and U.S. Patent Nos. 4,040,388, 4,469,047, and 4,593,646 to Miller, the entire contents of which are each. incorporated into this document. Such devices, as adapted to practice the present invention, generally comprise an injector containing the immunogenic composition, with the injector positioned to inject an egg loaded by the apparatus with the immunogenic composition. In addition, if desired, a sealing apparatus operatively associated with the injection apparatus may be provided to seal the hole in the egg after its injection.

In one embodiment, the apparatus for practicing the present invention may be as disclosed in U.S. Pat. No. 4,681,063 to Hebranke in U.S. Pat. No. 4,903,625 to Hebrank, the disclosures of which are incorporated herein by reference. This device comprises an injection apparatus for dispensing fluid substances for an egg plurality and a suction apparatus which fits, simultaneously suspends a plurality of individual eggs from its upward facing parts and cooperates with the injector to inject the eggs while the eggs are fitted by the suction device. Those skilled in the art will appreciate that this device can be adapted for injection into any part of the egg by adjusting the injector penetration depth as is known in the art. The present invention may also be practiced with the apparatus and methods described in US Patent No. 6,244,214 to Hebrank (the entire contents of which are incorporated by reference herein), wherein an apparatus for identifying the structure and or specific compartment within the egg is described. contact with a needle that has penetrated the eggshell, and methods for employing the apparatus for dispensing compositions for specific structures and / or compartments within an egg.

The appropriate volume of the immunogenic composition to be administered will depend on the size of the egg being treated, with ostrich eggs obviously being able to occupy more volume than chicken eggs. In particular embodiments, the immunogenic composition is administered in a volume of from about 10 to about 500.1000 or 2000 μΙ or more, including any number between 10 and 2000, even if not explicitly described herein, with illustrative volumes including 10, 20 , 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575 , 600, 626, 650, 675, 700, 725, 752, 775, 800, 850,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000μl. Other volumes suitable for distribution of the immunogenic composition can be readily determined by those skilled in the art.

In accordance with the particular embodiments of the present invention, eggs (i.e. embryonic birds) administered with immunogenic deposition are in the last half or last quarter of egg incubation (i.e. embryonic development). For example, for chicken eggs, the last half of incubation is from about the twelfth to the twentieth day of incubation (e.g., E12, E13, E14, E15, E16, E17, E17.5, E18, E18, 5, E19, E19.5 and / or E20), and the last quarter of the egg incubation is from approximately the fifteenth to the twentieth day of incubation (e.g., E15, E15.5, E16, E16.5, E17 , E17.5, E18, E18.5, E19, E19.5 and / or E20). In particular embodiments, the egg is administered with the immunogenic composition on the eighteenth (E18 or E18.5) or nineteenth (E19 or E19.5) day of egg incubation. In other embodiments, turkey eggs are administered with the immunogenic composition around the fourteenth to twenty-seventh day of incubation (E14, E15, E16, E17, E18, E19, E21, E21.5, E22, E22.5 , E23, E23.5, E24, E24.5, E25, E25.5, E26 and / or E27) around the twenty-first to the twenty-seventh day of incubation (eg E21, E21.5, E22, E22 , 5, E23, E23.5, E24, E24.5, E25, E25.2, E26, E26.5 and / or E27), or around the twenty-fifth (E25 or E25.5) incubation day. Those skilled in the art will appreciate that the present invention may be performed at any predetermined time in egg, provided that administration results in a desired immune response to the immunogenic composition, without undue levels of morbidity and / or mortality among treated patients.

Alternatively or additionally, the invention may be practiced to administer an immunogenic composition to a hatched bird, including newly hatched birds (i.e. approximately the first one, two and / or three days after hatching), adolescents, and / or adults. In certain embodiments, administration is within approximately the first six, five, four, three and / or two weeks after hatching and / or even within approximately the first week after hatching. According to one aspect of the invention, administration is within the first three weeks after hatching. In other embodiments, immunogenic attachment is administered to an egg (eg, in the first egg incubation room) and a booster is administered after hatching (eg within approximately the first one, two or three days or one, two or three weeks after hatching).

The methods of the invention can be distinguished from maternal vaccination approaches, in which older female birds (e.g., chickens around 10-15 weeks old) are vaccinated for the purpose of providing passive immunity to their offspring. Such birds are probably not previously untreated birds, ie they have already been exposed to Clostridium (eg Clostridium perfringens), and are not being immunized for the purpose of protecting the vaccinated bird, but instead to protect offspring by transfer. passive antibody.

The immunogenic compositions of the present invention may be administered to birds hatching by any suitable means.

Illustrative means are oral administration (e.g., in food or drinking water), intramuscular injection, subcutaneous injection, intravenous injection, intra-abdominal injection, eye drops and / or nasal spray. In addition, birds may be administered with the immunogenic compositions in a spray box, that is, a box in which birds are placed and exposed to a vaccine-containing vapor, or by spray-on.

The invention may be practiced to protect a bird from enteritenecrotic. By "protecting", "protecting" and "protection" and similar terms is meant any level of protection from necrotic enteritis that is of benefit to a patient population, such that there is a reduction in the incidence and / or severity of the disease between treated birds, whether in the form of decreased mortality, decreased lesions, improved body nope, improved feed conversion rates, or reduction of any other deleterious effects, regardless of whether the protection is partial or complete. Those skilled in the art will understand that protection can be assessed from a plurality of treated birds compared to untreated birds, even if individual treated birds are not protected. In particular embodiments, the invention provides a method of reducing the incidence of necrotic enteritis among a plurality of birds that are administered with the immunogenic compositions of the invention. Also provided is a method of reducing morbidity and / or mortality among a plurality of birds that are treated in accordance with the present invention.

By "initiating", "initiating" or "initiating" (and their grammatical variations), as used herein, is meant to initiate an immune response that is less than protective until a second tersion (booster) dose is given at a later time. By hatching. By "reducing", "reducing", "reducing" and "reducing" (and their grammatical variations), as used herein, is intended a decrease in the indicated parameter related to infection or disease (eg, infection, morbidity). , mortality, incidence of necrotic enteritis, similar injuries) of any value or benefit to the user (eg commercial value), eg a decrease of at least about 20%, 25%, 35%, 50% 75%, 80%, 85%, 90%, 95%, 97% or more compared to untreated birds.

The invention also provides methods of protecting birds from infection with the Clostridium species, which results in any level of protection that is of any benefit to a patient population, so that there is a reduction in the incidence and / or severity of Clostridium infection among treated birds.

The invention may also be practiced to induce an immune response to Clostridium. As used herein, the term "inducing" (or its grammatical variations) an immune response against Clostridium is intended to include agents that induce an immune response against the organism itself and / or toxins produced by the organism by passive transfer or immune response. Optionally, the immune response that is induced is a protective immune response, for example, in vaccination methods. Protection is not required if there is any other purpose to induce the immune response, for example for research purposes or to produce antibody for passive immunizations or as a reagent (for example to detect, isolate and / or identify Clostridium species).

As used herein, unless otherwise indicated, "C. perfringens" is intended to include C. perfringens type A and / or C. perfringens type C and / or any other type of C. perfringens that is implicated in etiology of necrotic enteritis in birds. In particular embodiments, the invention provides methods of protecting birds from C. perfringens type A and / or C. perfringens type C infection. The invention also provides methods of inducing an immune response against C. perfringens type A and / or C. perfringens type C. The different types of C.perfringens and their strains are well known in the art. See, for example, AMERICAN ASSOCIATION OF AVIAN PATHOLOGISTS, ALABORATORY MANUAL FOR THE ISOLATION AND IDENTIFICATION OF PATHOGENS (3rd ed. 1989).

The term "effective immunizing dose" as used herein, unless otherwise indicated, means an immunogenic composition dose sufficient to induce a protective immune response in the treated birds, which is greater than the inherent immunity of unimmunized birds. In the case of egg-treated poultry, an "effective immunization dose" indicates a sufficient dose to induce a protective immune response in egg-hatched birds that is greater than the inherent immunity of non-egg-immunized birds. . An effective immunizing dose in any particular context can be routinely determined using methods known in the art.

An "effective immunizing dose" may comprise one or more (for example, two or three) doses of the immunogenic composition to achieve the desired level of protection. Individual doses may be given in egg and / or after hatching.

As discussed above, it will be apparent to those skilled in the art that when treating a plurality of birds (such as in the production of commercial poultry), the efficacy of dose and / or immunogenic composition can be estimated by assessing the flocking effects on the flock as one all. In other words, an effective immunizing dose or an effective vaccine for the flock as a whole may, however, not induce an immune response and / or provide sufficient protection against disease in individual birds.

The terms "vaccination" or "immunization" are well understood by nature, and are used interchangeably herein. For example, the terms vaccination or immunization may be understood to be a process that enhances a patient's immune reaction to the antigen (providing an active immune response) and thus its ability to resist, overcome and / or recover from infection (ie. , an immune-protective response).

The terms "protective immunity" or "protective immune response" as used herein are intended to mean that the host animal prepares an active immune response to the immunogenic composition and / or that the immunogenic composition provides passive immunity, such that upon subsequent or subsequent exposure. As a provocation, the animal is able to resist or overcome infection and / or disease. Thus, a protective immune response will decrease the incidence of morbidity and / or mortality from subsequent exposure to the pathogen among treated birds.

An "active immune response" or "active immunity" is characterized by "participation of host tissues and cells after an encounter with the immunogen. It involves the differentiation and proliferation of immunocompetent cells in lymphorecticular tissues that result in antibody synthesis or the development of reactivity. mediated by cells, or both. " Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively established, an active immune response is prepared by the host upon exposure to immunogens by infection or vaccination. Active immunity can be contrasted with passive immunity, which is acquired through "transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a nonimmune host." Id.

Necrotic enteritis (NE) models for assessing the efficacy of vaccines and vaccination strategies are known in the art. For example, Hofacre et al. (2003, J. Appl Poult. Res. 12: 60-64) described a model in which chickens were fed a 26% fish meal soybean diet from day 0 to day 14 after hatching. Fishmeal was removed from the diet on day 14. Asaves were challenged with oral gavage coccidia on day 14, then daily from 17-19 with C. perfringens by oral gavage. Food conversion ratio, body weight, and bowel injury score were used to assess the presence and severity of enteritenecrotic in the challenged birds. NE lesions were assessed on day 22 or day 28 using a scale of O = none, 1 = mild, 2 = moderate and 3 = severe / severe. Other models may be used to evaluate vaccine efficacy and vaccine regimens, as known in the art. In certain embodiments of the present invention, administration of a composition of this invention (e.g., an effective amount of a composition of this invention) may result in a reduction of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. , 60, 70, 75, 80, 90, or 100% in intestinal lesions and / or changes in nosanimal body weight of this model or another model known or accepted in the art, compared to non-immunized or control animals.

In additional embodiments of this invention, the compositions and methods of this invention may be used to induce a bird antibody response that is at least greater than or equal to about 0.5 antitoxin unit / ml (e.g. at least about of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6 , 5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 AU antitoxin antibody per ml of bird antisera).

In additional embodiments where the compositions and methods of this invention are employed, the percentage of eggs of an egg population in which a composition of this invention is distributed to the embryo body may be from about 70% to about 100% (e.g., 70%). , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95 , 96, 97, 98, 99 or 100%) of the total number of eggs in the egg population to which the composition is administered.

The present invention also includes immunogenic compositions which induce an active and / or passive immune response against C. perfringens (including types A and / or C), and which may optionally be used to protect a bird against C. perfringens and / or to protect against necrotic enteritis, as described in more detail below.

The immunogenic compositions of the invention comprise, consist essentially of, and / or consist of (an) agent (s) that induce an immune response against Clostridium. The Clostridium immune agent may be a replicating antigen and / or a non-replicating antigen. The replicating and non-replicating antigens of this invention may be distributed in egg to the amnion, to the embryo, and / or to both the amnion and the embryo.

Further, the immunogenic compositions of the invention comprise, consist essentially of, or consist of, an effective immunizing dose of a Clostridium immunizing agent in a pharmaceutically acceptable carrier. In representative embodiments, immunogenic involvement may be formulated with Clostridium toxoids and / or bacterins. According to this embodiment, the optional immunogenic composition further comprises an adjuvant (see below). Ostoxoids are inactivated toxins, and may be derived from Clostridium toxins, including those derived from C. perfringens, including toxinalpha, beta toxin, beta2 toxin, enterotoxin, epsilon toxin, toxinote, kappa toxin, toxin lambda, and / or theta toxin; those derived from C. sordellii, including hemorrhagic toxin and / or lethal toxin; those derived from C. difficile, including toxin A (enterotoxin) and toxin B (cytopathic toxin); those derived from C. septicum, including alpha toxin, those derived from C. novyl, including alpha toxin and / or beta toxin, and / or those derived from C. botulinum, including type C toxin. Toxoids are known in the art and include, for example, treatment of the toxin with formaldehyde or heat (see, for example, Walker, (1992) Vaccine 10: 977-990). Bacterins are bacterial cell components and may be derived from a species of Clostridium, such as, for example, from C. perfringens types A and / or C. perfringenstipo C. C. perfringens toxoid vaccines are known in the art ( see, for example, U.S. Patent No. 4,292,306 to Zemlyakova). In other embodiments, the immunogenic composition comprises, consists essentially of, or consists of, a dead (i.e. non-replicating) Clostridium (i.e. bacterin) bacterium. optionally in a water-in-oil-in-water emulsion (see, for example, U.S. Patent No. 5,817,320 to Stone, which describes the egg immunization of avian embryos with oil emulsion vaccines, the entire contents of which are incorporated by reference herein. ), and / or a pharmaceutically acceptable carrier. In other embodiments, immunogenic composition comprises, consists essentially of, or consists of, dead Clostridium and an adjuvant (e.g., an aluminum adjuvant such as aluminum hydroxide, a saponin, such as Quil-A, including QuiIA QS21, or a oil, such as Freund's incomplete or incomplete), optionally in a water-in-water emulsion and / or a pharmaceutically acceptable carrier.

As a further alternative, the immunogenic composition comprises, essentially consists of, or consists of, a Clostridium immuno-replicating agent, for example live C. perfringens, which is generally a live attenuated (i.e., reduced virulence) C. ( See, for example, PCT Publication No. PCT WO 2005/053737, the entire contents of which are incorporated by reference herein, for teachings on the production of live attenuated bacteria for vaccine use.) Methods of producing attenuated bacteria are known in the art and include , without limitation: irradiation, chemical treatment, serial passage in culture, and the like. In certain embodiments of the invention, live Clostridium bacteria (e.g., C. perfringens) are administered in the presence of an agent that protects the patient from the pathological effects of the organism, for example by co-administering a neutralizing factor as described in US Patent. No. 6,440,408 to Thomae others, or interferon as described in US Patent No. 6,506,385 to Boston and others. Optionally, Clostridium and the neutralizing factor and / or ointerferon are administered in the same formulation. Additional examples of immunogenic compositions include compositions comprising, consisting essentially of, or consisting of, antitoxins (i.e. antibodies that provide passive immunity against Clostridiurrr alpha and / or beta toxins, see, for example, US Patent No. 6,719,267 to Carroll et al.), antigenic peptides which induce an immune response against Clostridium (including C. perfringens toxins, see for U.S. Patent Nos. 5,817,317 and 5,851,827 to Titball et al .; U.S. Patent No. 6,610,300 to Segers et al .; US Patent No. 5,695,956 to McCIane et al.), and recombinant vaccines comprising a carrier nucleic acid (e.g., a plasmid or virus) which releases a nucleic acid encoding antigenic peptide (s) or protein (s). (s) inducing an immune response against Clostridium.

In representative embodiments, the immunogenic composition comprises, consists essentially of, or consists of, a recombinant Clostridium alpha and / or beta toxin, for example, alpha toxins having amino acid sequence as shown in SEQ ID NO: 2 [natural size sequence 370] amino acids; GenBank Accession No. 1GYGB (Gl: 21730290), the coding sequence of which is provided in this document as SEQ ID NO: 1], SEQ ID NO: 4 (Cpa247-370; amino acids 247-370 of SEQ ID NO: 2; whose sequence provided in this document as SEQ ID NO: 3), SEQ ID NO: 6 (amino acids 1-278 of SEQ ID NO: 2; whose coding sequence is provided in this document as SEQ ID NO: 5), SEQ ID NO: 8 ( Amino acids 261-300 of SEQ ID NO: 2, the coding sequence of which is provided herein as SEQ ID NO: 7), as described herein and / or, for example, in US Patent Nos. 5,817,317 and 5,851. 827 for other Titballe, or SEQ ID NO: 10 [life-size sequence of 398 amino acids, whose coding sequence is provided in this document as SEQ ID NO: 9]. In some embodiments, the toxin of the present invention is an immunogenic composition comprising amino acids 1-278 (SEQ ID NO: 6) of the 370 amino acid sequence (SEQID NO: 2) of the C. perfringens alpha toxin.

Additional examples of toxins (including their immunogenic fragments) that may be used in the compositions and methods of this invention include, but are not limited to, C toxins. perfringens [e.g., alpha toxin, Accession number CAA35186 (Saint-Joanis et al. Mol. Gen. Genet. 219 (3): 453-460 (1989) 0; toxin beta, Accession number CAA58246 (Steinthorsdottir et al. FEMS Microbiol Lett. 130 (2-3): 273-278 (1995)); beta 2 toxin, Accession No. NP_150010 (Shimizu et al. Proc. NatL Acad. Sci. USA 99 (2): 996-1001 (2002)) ; enterotoxin Accession Number BAE79112 (Miyamoto et al. J. Bacteriol.188 (4): 1585-1598 (2006)); epsilon toxin Accession Number AAA23236 (Havard et al. FEMS Microbiol. Lett. 97: 77-82 ( 1992); iota toxin, Accession No. CAA51959 (Perelle et al., Infect. Immun. 61 (12): 5147-5156 (1993)); Hood toxin, Accession No. NP_561089, Shimizu et al Proc.NatL Acad. Sci. USA 99 (2): 996-1001 (2002)); lambda toxin, Accession Number CAA35187 (Saint-Joanis et al. Mol. Gen. Genet. 219 (3): 453-460 (1989)); and theta toxin, Accession Number NP_561079 (Shimizu et al. Proc.Natl. Acad. Sci USA 99 (2): 996-1001 (2002))], C. difficile toxins [e.g. toxin A Accession Number A37052 (Wren et al. FEMSMicrobiol. Lett 70: 1-6 (1990)); and toxin B, Accession number CAA43299 (vonEichel-Streiber et al. Mol. Gen. Genet. 233 (1-2): 260-268 (1992))], C. septicum toxins [toxin alfa, Accession number AAB32892 (Ballard and others Infect Immun. 63 (1): 340-344 (1995))] and C. novyi toxins [e.g., toxin alfa, Accession Number AAB27213 (Bali et al. Infect. Immun.61 (7): 2912- 2918 (1993))].

The terms "toxin" "alpha toxin", "beta toxin", "epsilon toxin" (or a similar term), etc., as used herein, include life-size toxin as well as antigenic or immunogenic peptides or antigenic variants. or immunogenic (e.g., attenuated) of them that induce an immune response (optionally a protective immune response) against Clostridium in the patient. In particular embodiments, an antigenic peptide comprises at least about 6, 8, 10, 12, 15, 18, 20, 25, 30, 50, 75 or 100 or more life-sized contiguous datoxin amino acids (see, for example, the sequence natural-sized alpha toxin as shown in SEQ ID NO: 2 in US Pat. Nos. 5,817,317 and 5,851,827 and SEQ ID NO: 2 herein).

It is also understood that the immunogenic fragments of this invention may be combined in any order or quantity. For example, fragment 1-10 may be combined with fragment 10-20 to produce an amino acid fragment 1-20. As another example, fragment 1-20 may be combined with fragment 50-60 to produce a single fragment of this invention having 31 amino acids (AA 10-20 and AA50-60). Also fragments may be present in multiple numbers and in any combination in a fragment of this invention. Thus, for example, fragment 1-150 may be combined with a second fragment 1-150 and / or combined with fragment 400-500 to produce a fragment. of this invention.

In some embodiments, an ouimmunogenic antigen fragment of a Clostridium toxin of this invention may comprise, consisting essentially of, and / or consisting of the terminal amino domain of the C. perfringens alpha toxin (amino acids 1-246 of SEQID NO: 2), domain terminal carboxy of the C. perfringens alpha toxin (amino acids 256-370 of SEQ ID NO: 2) and / or fragment between these domains (amino acids 247-255 of SEQ ID NO: 2) in any combination with any amount of overlap in the sequence. amino acid that results in a fragment having immunogenic activity. This language is intended to include all possible peptide and toxin fragments as explicitly described herein (for example, any peptide or fragment comprising at least about 6, 8, 10, 12, 15,18, 20, 25, 30, 50 75 or 100 or more contiguous amino acids of the life-size alpha toxin sequence as shown in SEQ ID NO: 2 at 30 US Patent Nos. 5,817,317 and 5,851,827 and SEQ ID NO: 2 and SEQ IDNO: 10 herein ). In particular embodiments, the antigenic peptide does not have an amino acid sequence having the activity of hydrolyzing phospholipase C and / or sphingomyelin (for example, an antigenic alpha toxin peptide may not have amino acids 1-240). The position of some C. perfringens alpha toxin epitopes has been determined (see, for example, Logan et al., (1992) Infection and Immunity 59: 4338-4382, whose entire contents are incorporated by reference to the teachings of alpha toxin epitopes).

Additional examples of recombinant Clostridium toxins that may be employed in the methods of this invention include, but are not limited to, a Clostridium perfringens beta toxin or an immunogenic fragment thereof, where the beta toxin has the amino acid sequence as described in SEQ ID NO: 11. The beta toxin of SEQ ID NO: 11 may additionally comprise a mutation at amino acid 62, 182, 197 or one of the regions between amino acid numbers 80-103, 145-147,281-291, 295-299 or downstream of amino acid position 292 (as described in US Patent No. 6,610,300, the entire contents of which are incorporated by reference herein), whereby the resulting toxin fragment has immunogenic activity.

The nucleic acid and amino acid sequences of C. perfringens alpha and beta dastoxins are known in the art, see, for example, GenBank Access N- DQ202275; NP_560952; NC_003366; AY823400; AY277724; AF204209; X17300; X13608; L43548; L43547; L77965 and L13198. See also, Sheedy et al., Highly Conserved Alpha-Toxin Sequences of Avian Isolates of Clostridium perfringens, J. Clin.Microbioi 42: 1345-1347 (2004), which presents an analysis of the toxin alpha sequence of 25 strains of C. perfringens derived from chickens.

In the additional embodiments of this invention, the Clostridium toxin may be a C. perfringens epsilon (ε) toxin having an amino acid sequence as described in SEQ ID NO: 12 (328 amino acids; or SEQ ID NO: 13. In additional embodiments, the toxin may comprise the amino acid sequence of SEQ ID NO: 13, wherein residue 2 is a proline, as described in US Patent No. 6,403,094, the entire contents of which are incorporated by reference herein. In certain embodiments, the present invention provides a method of immunizing An avian patient against Clostridium infection, comprising administering to the avian patient an effective immunizing dose of a Clostridium bacterin toxoid composition by injection into an egg during the final incubation room.The methods of this invention may further comprise the step of administering a booster dose of bacterial toxoid composition of Clostridium to the patient after hatching. The Clostridium species of this invention may include but is not limited to Clostridium perfringens. In the particular embodiments of this invention, the composition may comprise a Vision CD® vaccine. In particular embodiments where the patient is a chicken, the bacterin-toxoid composition may be administered to the amniotic fluid via a 20g, 2.54cm (1 inch) needle on day 18 of incubation or a 22g, 2.54 needle. inches (1 inch) on day 18 of incubation.

In additional embodiments of this invention, there are provided methods of immunizing an avian patient against Clostridium infection, comprising administering to the avian patient an immunizing-effective dose of a recombinant toxin, or immunogenic fragment thereof, of Clostridium by injection into an egg during the final fourth incubation. . In some embodiments, these methods may further comprise the step of administering a booster dose of the recombinant toxin, or immunogenic fragment thereof, to the avian patient after hatching. In particular embodiments, the composition employed in these methods may comprise an adjuvant, which may be incomplete Freund's Quil Aeo adjuvant. In modalities where the patient is a chicken, bacterin toxoid may be administered to the body of the embryo via a 23g, 3.125 cm (1.25 inch) needle during the 19th day of incubation.

In still further embodiments, when the patient is a small picture, a toxin or immunogenic fragment thereof of this invention may be administered to the embryo's body via a 20 g, 3.81 centimeter (1.5 inch) needle during the day of incubation. Also, the dosage range of a toxin (e.g., an alpha toxin) or fragment thereof and / or other subunit protein or glycoprotein or other type of molecular molecule used as a vaccine of this invention may be from about 1 μg to about 1000 μg per dose, with an illustrative range of about 55 μg to about 60 μg per dose. For the compositions of this invention comprising inactivated virus, the virus concentration per dose can be from about 1012 EID50 / TCID50 to about 1012 EID50 / TCID50 (EID = infectious dose of egg; TCID = infectious dose of tissue culture). In modalities comprising activated virus, the pordose virus concentration may be about 10 ° · 1 EID5 ° / TCID5to about 1012 EID5 ° / TCID5 °. In particular embodiments of this invention, the toxin of this invention comprises, consists essentially of, and / or consists of, the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, including any combination of these.

As contemplated herein, in some embodiments of the present invention, the composition of this invention further comprises an adjuvant which, in particular embodiments, may be an adjuvant such as an aluminum derived adjuvant (e.g. aluminum hydroxide), a saponin (e.g. Quil-A, including QuiIA QS21), or an oil (such as Freund Complete or Incomplete adjuvant) in any combination. Additional examples of adjuvants that may be employed in any of the inventive methods described herein are provided herein.

In representative embodiments, the immunogenic composition of this invention comprises, consists of, or essentially consists of, a C. perfringens enterotoxin (CPE), beta-2 toxin, epsilon toxin, kappa toxin, lambda toxin, theta toxin, and / or iota toxin. optionally in addition to a C. perfringens alpha and / or beta toxin.

In other illustrative embodiments, the immunogenic composition comprises, consists essentially of, or consists of, an outoxoid / bacterin toxoid. The bacterin may be a C. perfringens type A and / or type C bacterin. For example, an illustrative immunogenic composition comprises, consists essentially of, or consists of, a C. perfringens type A bacterial toxoid alpha and an optionally the immunogenic composition additionally. comprises an adjuvant, such as an aluminum-derived adjuvant (e.g., aluminum hydroxide), an aponine (e.g., Quil-A, including QuiIA QS21), or an oil (such as Freund's Complete or Incomplete's adjuvant).

A representative immunogenic composition of the invention comprises, consists essentially of, or consists of, an effective immunizing dose of a C. perfringens immunizing agent in a water-in-oil emulsion (see, for example, US Patent No. 25,817,320 to Stone), optionally in a pharmaceutically acceptable carrier.

The immunogenic composition may optionally comprise two or more agents that induce an immune response against C. perfringens (e.g., any combination of the above described agents).

In particular embodiments, the agent that induces an immune response against C. perfringens (e.g., an attenuated toxoid, bacterin, C. perfringens, and / or toxin and the like) is a bird derived C. perfringens strain, optionally a chicken derivative. .

As used herein, the term "consists essentially of" (and grammatical variants) means that the immunogenic composition comprises no material immunogenic agent other than those indicated herein. The term "essentially consists of" does not exclude the presence of other components, such as adjuvants, immunomodulators, and the like.

By "pharmaceutically acceptable" is meant a material which is not biologically, or otherwise undesirable, that is, the material may be administered to a patient without causing appreciable undesirable biological effects. Thus, such pharmaceutical composition may be used, for example, to prepare compositions for immunization. Physiologically and pharmaceutically acceptable vehicles may contain other compounds, including but not limited to stabilizers, salts, buffers, adjuvants and / or preservatives (e.g., antibacterial, antifungal and antiviral agents) as are known in the art. The pharmaceutically acceptable carrier need not be sterile, although it is typically for egg administration to avian embryos.

In particular embodiments, the immunogenic composition further comprises an immune stimulant. Alternatively, the immune stimulant may be administered to the patient in a separate formulation. Immune stimulants that may be used in the present methods include, but are not limited to, cytokines, growth factors, chemokines, lymphocyte cell culture supernatants, monocytes, or lymphoid organ cells, cell preparations, or cell extracts (e.g., fixed preparations of Staphylococcusaureus or lipopolysaccharides), mitogens, or adjuvants, including low molecular weight pharmaceutical substances. Immune stimulants may be administered to egg at any time during incubation. Optionally, the immune stimulant and agent that induces an immune response against C. perfringens are concurrently administered, an optional method in the same formulation.

As used herein, the word "concurrently" means sufficiently close in time to produce a combined effect (ie, concurrently may be simultaneously, or may be two or more events occurring within a short time before and / or after each other).

Any suitable vaccine adjuvant may be used in accordance with the present invention, including chemical immunostimulants and polypeptides that enhance the immune system response to antigens. Adjuvants include, but are not limited to, an aluminum-derived adjuvant (e.g. aluminum), an aluminum phosphate, vegetable and animal oils (eg from Incomplete or complete Freund), saponin (eg Quil-A, including QuiIA QS21), Spur® (Intervet), similar. Representative adjuvants of this invention include, but are not limited to, an aluminum salt, such as aluminum hydroxide gel (alum), aluminum phosphate, or algamulin, but may also be calcium or magnesium salt, iron or mineral gels. zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationic or anionically derived polysaccharides, or polyphosphaenes, ousaponins, such as Quil-A, or oil emulsions, such as water in oil and water in oil in water or Freund complete or incomplete or any combination thereof.

The immunogenic composition may optionally contain one or more stabilizers. Any suitable stabilizer may be used, including carbohydrates such as sorbitol, mannitol, starch, asacarose, dextrin, or glucose; proteins such as albumin or acasein; and buffers such as alkali metal phosphate and the like.

It is often convenient to immunize a bird against multiple diseases in a single course of treatment. Thus, in particular embodiments, the immunogenic composition comprises one or more additional agents that induce an immune response against other avian (e.g. viral, bacterial or fungal) pathogens, optionally immunizing agents that produce a protective immune response. For example, the immunogenic composition may further comprise an immunizing agent against coccidiosis (ie, Eimeria), bursal infectious disease, Marek's disease, Newcastle disease, avian influenza, avian reovirus, avian metapneumovirus, avian adenovirus, infectious bronchitis, Salmonella spp., Camplyobacter spp., a Pasteurella spp., Hemophilus paragallinarum and / or Mycoplasma spp. Avian vaccines suitable for use in egg or after hatching are known in the art and are commercially available (e.g., Bursaplex® avacin for bursal disease; Newplex® vaccine for Newcastle disease, and Inovocox® for coccidiosis vaccine). Ambrex, Inc., and Marek's HVT-SB-1 vaccine for Marek's disease, available from Merial). Immunogenic compositions comprising vaccine images against both coccidiosis (i.e. Eimeria) and necrotic aortaitis (i.e. C. perfringens) are particularly advantageous because exposure to Eimeria is known to increase the susceptibility of birds to necrotic enteritis due to gastrointestinal disturbance. .

Thus, as a further aspect, the invention includes methods of administering an immunogenic composition comprising, consisting essentially of, or consisting of, an effective immunizing dose of a C. perfringens immunizing agent and an effective immunizing dose of an immunizing agent providing protection against one or more other avian diseases (as described above). Multiple immunizing agents may be provided in a single formulation or may be administered simultaneously or sequentially in any order in separate formulations. As discussed above, this aspect of the invention is particularly suitable for co-administration of decoccidiosis and necrotic enteritis vaccines.

In another representative embodiment, the avian patient is first immunized against necrotic enteritis and is then immunized against coccidiosis or vice versa. Both immunizations may occur in young, both may occur after hatching, or one may be in egg and one after hatching. For example, in one illustrative embodiment, the patient is immunized against egg coccidiosis and then immunized against necrotic eenteritis after hatching.

The invention may also be practiced for administering an immunizing agent of C. perfringens to egg or after hatching in conjunction with "egg feeding" (see U.S. Patent No. 6,592,878; incorporated herein by reference) from the patient. For example, according to certain embodiments, a C. perfringens immunizing agent and an enteral nutrient and / or modulator formulation are administered to an avian egg patient, optionally by an ammonium distribution. Optionally, vaccines against other infectious agents are administered in egg and / or after hatching as well (as described above). The C. perfringens immunizing agent and nutrient formulation and / or the enteric modulator may be administered simultaneously, in the same or separate compositions, and / or may be administered sequentially in any order.

Additional embodiments of the present invention may include a composition comprising an antigen selected from the group consisting of a C. perfringens alpha toxoid, an antigenic fragment of a C. perfringens alpha toxoid, an inactive antigenic fragment of a C. perfringens alpha toxin, and any combination thereof; where one or more doses of from about 0.1 to about 1.0 ml (e.g. 0.1, 0.2, 0,3,0.4, 0.5, 0.6, 0.7, 0 8, 0.9, or 1.0) per dose of the composition is sufficient to induce at least 0.5 antitoxin (AU) unit of antitoxinalpha antibody per ml of antisera from a bird (e.g., chicken) vaccinated with vaccine. In some embodiments, the composition may induce at least about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 , 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5 or 10 AU of antitoxin antibody per ml of daave antisera.

As used herein, an "antitoxin unit" or "A.U." Antitoxin antibody level per ml antisera (which can be used interchangeably with "Antitoxin Neutralization Test" units or "TNT" units) is defined by the ability of the sera to counteract the toxic effects of a toxin in a mouse bioassay. . In this test, a known amount of toxin, established by international standards as is known in the art, is mixed with serial dilutions of serum from vaccinated animals. The mixture is incubated one hour at room temperature and then injected intravenously into the mice. The mice will survive if the toxin is completely neutralized by the sera, otherwise they die. The antitoxin units or titer is determined as the reciprocal of the highest dilution of sera that neutralized atoxin.

In additional embodiments, the composition may comprise an antigen in a cell-free preparation. In other embodiments, the antigen may be an alpha toxoid in a C.perfringens alpha toxoid supernatant. In certain embodiments, the composition may comprise, consist essentially of, and / or consist of, an antigen which may be C. perfringens Type A alpha toxoid and / or a C. perfringens Type C alpha toxoid. In some embodiments, the The composition of this invention may comprise C. perfringens beta toxin, C. perfringens beta 2 toxin, C. perfringens enterotoxin, C. perfringens epsilon toxin, C. perfringens iota toxin, C. cape toxin. perfringens, C. perfringens toxin Iambda, C. perfringens theta toxin, C. sordelli haemorrhagic toxin, C. sordeilii lethal toxin, C.diffici toxin A, C. difficile toxin B, C. septicum alpha toxin, C. novyi alfade toxin, C. novyi beta toxin and / or any combination thereof. Such a composition may further comprise essentially consisting of, and / or consisting of, one or more viral antigens, one or more bacterial antigens, and / or one or more parasitic antigens, as described herein.

The present invention is further explained in the following non-limiting examples. In these Examples, "μΙ_" means microliters,> g "means micrograms," mL "means milliliters," cm3 "means cubic centimeters," mm "means millimeters," mM "means concentration in millimoles," mg "means milligrams," SC "means degrees Celsius and E18 and E19 mean embryonic days 18 and 19, respectively.

EXAMPLES

Example I. Immune response following egg vaccination with Clostridium perfringens Type D vaccines (Siteguard G and Vision CD).

Experimental Project

Eggs with the chicks were manually injected with commercially available toxoid vaccines (Siteguard®) and debacterin toxoid (Vision® CD® vaccine) from Ciostridium perfringens. Birds hatching were developed to measure antibody responses. Injection site evaluation was performed. On day O (hatching), the selection treatment groups received a post-hatching vaccination. All birds were housed in cages (5 birds / cage). Cadagaiola was supplied with a diet of Normal Broiler Starter. On day 14, the birds were switched to Broiler Grower Feed. The birds were bled and the serum was tested for an antibody response by the serum toxin neutralization assay.Materials and MethodsInjection Material (Siteguard® G):

Siteguard® G (unknown adjuvant) is a C. perfringens type C and D detoxoid vaccine produced by Schering-Plow. It protects sheep and cattle from diseases caused by Type D toxins. For vaccination, 4.0 mL (cattle) or 2.0 mL (sheep) of the vaccine are administered subcutaneously (SQ) or intramuscularly (IM). Booster vaccinations are administered three to four weeks after initial avaccination and annually.

Injection Material (Vision® CD®):

Vision® CD® (with patented 'Spur®' adjuvant) is a C. perfringens CeD-type bacterin toxoid vaccine produced by Intervet. It protects cattle, sheep, and goats from C. perfringens CeD-type enterotoxemia. For vaccination, 2.0 mL of vaccination is administered subcutaneously to the animal (cattle, sheep, or goats) .Three to four weeks after the initial vaccination, the animal receives an additional 2.0 mL (SQ) and is vaccinated again annually. after this.

Injection Protocol:

In E19, eggs with the chicks were injected with the test materials targeting the amniotic fluid or embryo body of each egg. In addition, on day 0 after hatching, some egg-injected and non-egg-injected treatment groups received a vaccination or immunization by strengthening test materials. For vaccination or booster immunization after hatching, 0.5 mL of vaccine was administered by subcutaneous injection into the back of the neck.

Injection Site:

At the time of injection, eggs intended for injection site evaluation were injected with dye. The eggs were then euthanized for autopsy to evaluate the injection site. The injection site was analyzed by the chi-square of the likelihood ratio (Table 1).

On days 7, 14, 21, and 28 after hatching, blood was collected from each individual bird and pooled into individual vacutainers (per treatment group). On days 7, 14 and 21; <0.5 ml_ of blood was collected via the wing or the jugular. On day 28,> 0.5 mL of blood was collected via cardiac perforation. The blood was then incubated at room temperature for one hour. Then the blood samples were placed in a table top centrifuge at 2400 RPM for 10 minutes. Once centrifugation was complete, serum was removed from each blood sample and stored in a 96-well storage plate (2-8-C or -70-C) for future evaluations of the immune response.

Clostridium perfringens TypeC Toxin (beta) Neutralization Test in Mice: Sample Preparation for Inoculation of MouseA. Materials

1. Clostridium perfringens Toxin (beta) Type C - Lot No. CVBLIRP513 (04)

2. Ciostridium perfringens Type C Antitoxin (beta) - Lot No. CVBLIRP486 Receipt

3. Diluent - 1% Peptone, 0.25% NaCl pH 7.2, - Batch No. BBL051006, ref. NB - NB 140 p. 87

4. Chicken Serum Samples - EMHE1381, No. 140 p. 80

The. Sample Nos. 7, 8, 9, 10A, 10B, 11A, 11B, 12A, 12B, 13A, 14A, 15A

5. 3 ml and 1.8 ml sterile vials.

B. Methods:

1. Dilute standard beta antitoxin 1:50 in diluent, total 10 ml.

The. Thaw antitoxin beta at temp. environment.

B. Mix 200 μl of beta antitoxin and 9.8 ml of diluent = 1: 50c. Keep on ice.

2. Dilute beta 1: 120 toxin in diluent, total 12 ml.

The. Thaw the beta toxin at temp. environment.

B. Mix 200 μΙ beta toxin and 1.8 ml diluent = 1:10

ç. Mix 1 ml of 1:10 diluted beta toxin with 11 ml of diluent = 1: 120

d. Keep on ice.

3. Prepare control sample L0.

The. Mix 0.5 ml beta toxin (1: 120 dilution) with 0.5 ml dediluent.

B. Add 1 ml of antitoxin beta (1:50 dilution).

ç. Mix and incubate at room temperature for 1 hour.

d. Keep samples on ice.

4. Prepare the L + control sample.

The. Mix 0.8 ml of beta toxin (1: 120 dilution) with 0.2 ml of

diluent.

B. Add 1 ml of antitoxin beta (1:50 dilution).

ç. Mix and incubate at room temperature for 1 hour.

d. Keep samples on ice.

5. Prepare the 12 test serum samples.

The. Mix 3.25 ml beta toxin (1: 120 dilution) with 3.25 ml dediluent.

B. To each of the twelve 1.8 ml tubes add 0.5 ml of toxin.

from step 5.a.

ç. Label each tube with the sample number and the number of the

treatment group.

d. Add 0.5 ml of each undiluted chicken serum to the appropriately labeled tube.

and. Mix and incubate at room temperature for 1 hour.

f. Store samples on ice.

6. All unused samples are stored at 2-7sC. Pre-Study Activities

Seventy-eight female Swiss (CD-1) white mice (16-20 grams body weight) were purchased for use in the study. The mice were shipped from the seller (Charles River Laboratories) and transported to the Clinical Test Facility.

The mice were housed in cages, placing 2 mice per cage for the chicken serum groups and 4 cages of 5 mice per cage for the control groups. The mice were maintained for a 5 day acclimation period prior to the start of the study on Day 0. The mice were housed and cared for according to standard operating procedures and fed a standard dietary diet and served ad libitum water.

The mice were examined for health and normal appearance and tested by listing seventy-six mice. Two mice were not enrolled in the study and were euthanized. Each mouse was injected intravenously (IV) with a 26g χ3 / 8 needle into the tail vein, according to the treatment groups described under STUDY DESIGN. Mice were monitored twice daily for signs of shock, pain or suffering, as evidenced by the following:

Lethargy

Shrinkage

Hardhaired / tanned skinArched posture

Ataxia

Anorexia or inability to reach food and water.

Dying mice were euthanized via excessive CO2 dosing according to standard operating procedures. Day 1 (approximately 24 hours after inoculation)

The mice were observed and the number of mortalities

registered.RESULTS

A positive specific antibody response was detected in our egg-vaccinated poultry (target embryo body) with a commercial C. perfringens bacterin toxoid vaccine, with and without booster after hatching, using the pore toxin neutralization assay. These data indicate that administration of a C. perfringes bacterin toxoid vaccine in egg produces partial protection and egg administration followed by a booster after hatching gives full protection against C. perfringens (Table 2).

EXAMPLE II. Immune response following egg vaccination with an experimental vaccine preparation containing recombinant toxin alpha

Experimental Project

The above study was conducted to determine their humoral (antibody) immune response could be detected in chicks after egg vaccination, egg + after hatching or after hatching with recombinant Clostridium perfringens atoxin alfa (SEQ ID NO: 6 ) (provided by Dr. Glenn Songer, Department of Veterinary Science and Microbiology, University of Arizona), with Incomplete Freund's Adjuvant and Quil-A. The immunization strategy included targeting the egg embryo body at E18, as well as a post-hatch vaccination on day 7. Antibody response was assessed at 28 days of age.

In E18, chick eggs were manually injected in new with control materials (Quil A; Accurate Chemical & Scientific Corporation, Product No. AP04991, Adjuvant Grade, 'Batch No. L77-238) emulsified with Incomplete Freund's Adjuvant (IFA; Rockland, Lot # 216235) or C. perfringens recombinant alpha toxin (55 or 60 μg / dose) with Quil A + IFA adjuvant (13 or 15 μg / dose). Injection site evaluation was performed by dye injection. On day 0 (hatching), the birds were housed in cage units (5 birds / cage). The birds received Normal Broiler Starter. Additionally, some birds were vaccinated nodia 7 or 17 according to treatment (0.2 mL subcutaneous injection vaccine at the back of the neck). Bird sera were then evaluated for western blot specific antibody response.

Materials and methods

Injection:

In E18, chick eggs were injected with the test materials (Clostridium perfringes alpha toxin + vaccine adjuvant) to cleanse the embryo body of each egg. In addition, on day 7 or day 17 after hatching, some non-injected and egg-injected treatment groups received a vaccination (Table 3). Injection site (SOI):

At the time of injection, the eggs intended for SOI evaluation were injected with dye. The eggs were then euthanized and necropsied for the assessment of SOI (Table 4).

Serum Collection:

On days 7, 14, 21, and 28 after hatching, blood was collected from each individual bird and pooled into individual vacutainers. On days 7, 14, and 21, <0.5 ml_ of blood was collected via the wing or jugular vein. On day 28,> 0.5 mL of blood was collected via cardiac perforation. The blood was then incubated at room temperature for 1 hour. Blood samples were centrifuged and the serum was removed and stored in a 96-well storage plate (2-8 ° C or -70 ° C) for immune response evaluation.

Western blot test:

SDS plated gel electrophoresis was performed according to the Laemmli method (Nature 227: 680-685 (1970)) as described by O'Farrell (J. Biol. Chem. 250: 4007-4021 (1975), second dimension ) using a 10% acrylamide plate gel (125 mm length X 150 mm width X 0.75 mm thickness) overlaid with a 25 mm stacking gel. Electrophoresis was performed at 12 mAmp for about 3.5 hours or until the bromophenol blue front had migrated to the end of the plate gels. Following plaque gel electrophoresis, the spotting gel was placed in transfer buffer (12.5 mM Tris, pH 8.8, 96mM glycine, 20% MeOH) and transmanced onto a PVDF membrane overnight at room temperature. 200 mA and approximately 100 volts / two gels. PVDF membrane was then stained with Coomassie blue and dried between sheets of filter paper.

The PVDF membrane was stained with Coomassie R-250 Bright Blue and scanned on a desktop computer before and after the cutout in individual paths. Each stain run was placed in a separate container and blocked for two hours in 5% non-fat dry milk in Tween-20 Tris (TTBS) buffered saline and rinsed in TTBS. The spots were then incubated in anti-primer (diluted 1: 100 in 2% non-fat dry milk in TTBS) for night and rinsed 3X10 minutes in TTBS.

The blot 1 lane (positive control) was then placed on the secondary antibody [goat anti-lgG-rabbit HRP (Sigma, Cat. No. A-5420 and Batch η2 034K4858), diluted 1: 5,000 in 2% NFDM in TTBS ] for two hours, rinsed 3 X 10 minutes in TTBS, treated with ECL and exposed to x-ray film.

The remaining blot strips were then individually placed in secondary antibody [anti-chicken-HRP IgG in rabbit (Bethyl, Cat. Η2 A30-107P and Batch η2 A30-107P-3), diluted 1: 2,000 in 2% non-fat dry milk. % in TTBS] for two hours, rinsed 3 X 10 minutes in TTBS, treated with ECL and exposed to x-ray film.

The results of the western blotting studies are described in

Table 5

Results

A specific antibody response was detected in the egg-vaccinated poultry (targeted embryo body) with the Quil-A and IFA adjuvanted alpha-recombinant toxin, with or without a booster after hatching. These data demonstrate that egg vaccination with a recombinant alpha toxin can produce an immune response against the alpha deC toxin. perfringens in chicks. EXAMPLE III.

A commercially available inactivated oil emulsion vaccine for Newcastle disease was purchased from Maine Biological Laboratories. The vaccine was administered in egg at E18 via the amniotic fluid route or in egg at E19 via the embryo body route. Site-directed administration to the amniotic fluid and body of the embryo was confirmed by conducting an analysis of the injection site using E18 or E19 staining. Directed administration to the amniotic fluid site was performed using a non-pointed needle (Group 2). Locally directed administration to the body of the embryo was performed using a 3.125 cm (1.25 inch) sharp needle (Group 3). Blood serum was collected at 14, 21 and 28 years of age and tested for Newcastle disease virus (NDV) specific antibodies using ELISA (Idexx, Inc.). Different individual birds were bled in a blood collection caddy.

Analysis of the injection site at E18 indicated that 22/24 eggs were injected into the amniotic fluid, 22/24 into the allantoic fluid, and 0/24 the embryo's body. Analysis of the injection site at E19 indicated that 7/1010 were injected into the body of the embryo and 3/10 eggs were injected into amniotic fluid. Table 6 shows the antibody response to Newcastle disease virus following chicken egg vaccination. Table 7 shows the hatching percentage data.

The study demonstrates that the developing embryo's immune response is strongly influenced by the new administration site of the Newcastle disease oil emulsion inactivated vaccine (Table 6). Embryos vaccinated in the amniotic fluid surrounding the embryo body did not respond with Newcastle disease-specific antibodies. On the other hand, embryos vaccinated directly into the body of the embryo responded with a strong antibody response that increased with age up to 28 days. A total of 34 birds were bled during the course of this study and 26/34 were positive for Newcastle disease virus antibodies (Table 6). 26/34 is 76.5%, which is very similar to the injection site study where 70% of the E19 immunized embryos were injected into the body of the embryo. The hatching percentage was within normal ranges for the treated and untreated groups (Table 7).

Example IV.

A commercially available inactivated oil emulsion vaccine for Newcastle disease was purchased from Maine Biological Laboratories. The vaccine was administered in egg at E19 via the embryo or subcutaneous hatching route. Directed administration to the site of the embryo's body was done using a sharp 3.175 cm (1.25 inch) needle (Group 3). Vaccination on hatching day was done by injecting the subcutaneous vaccine into the nape of newly hatched egg chicks (Group 2). Blood serum was collected on day 21 and tested for NDV specific antibodies using ELISA (Idexx, Inc.). Results are shown in Table 8.

The data presented in Table 8 show that embryos vaccinated in the body with the Newcastle disease oil emulsion vaccine responded, as well as chicks vaccinated via the standard hatching day. The hatching percentage was within normal ranges for the treated and untreated groups (Table 9).

The data presented in examples III and IV above show that the embryo's body hit is necessary to stimulate an active immune response to an inactivated antigen (in this case, the Newcastle disease virus). These data also indicate that embryos are not negatively affected by injection into the body of the embryo with an inactivated antigen in an oil emulsion adjuvant.

Egg injection (before hatching) into the body of the embryo can be done manually using a syringe and needle, or by an automation injection device also using needles. In the examples given herein, the syringe and needle were used manually to apply the vaccine to the embryo's body or to the amniotic fluid (Example III) that surrounds the embryo's body. For injection into the embryo body, the needle was inserted through a hole in the shell at the end of the egg air cell. The inserted needle passed through the air cell membrane, membranes and allantoic fluid and finally into the amnion cavity where the embryo body resides. Next, the needle penetrated the body of the embryo and the vaccine was deposited. Injections into embryo bodies can occur at various sites within the embryo body and include subcutaneous, intradermal, intravenous, intramuscular and intra-abdominal deposition of the vaccine, as well as any combination of these sites. In addition, injections into the body of the embryo may occur on the head, neck, shoulder, wing, back, chest or leg, including any combinations. Injection into the body of the embryo does not include adherence to the exclusive vaccine in the air cell, allantoic cavity, amniotic fluid or albumin.

Injection into the body of the egg embryo can be done using a needle of a length ranging from 1.905 cm to 10.16 centimeters (3A inch to 4 inch) and scales ranging from 15 to 28. Needle tips can range from very sharp (hypodermic). ) not even pointed.

In Examples III and IV, the vaccine for the Newcastle disease virus was used as the model antigen. However, any properly formulated oil emulsion vaccine with sufficient antigenic mass would be expected to be similar to the vaccine for Newcastlet's disease test. Therefore, inactivated vaccines for infectious bursal disease, avian influenza, infectious bronchitis, depot infectious anemia virus, laryngotracheitis, avian reovirus, adenovirus, rotavirus, oastrovirus, inclusion body hepatitis, fall, Escherichia coli, Mycoplasma spp., Salmonella spp., Campylobacter spp., Clostridium spp., Haemophilus spp., Pasteurellaspp. may be distributed in an egg directly to the embryo's body according to the methods described herein. Vaccines made from these agents may be whole cell or subunit. Vaccines made from these agents may be conventionally produced by growth, eggs or tissue culture and / or may be produced by recombinant means according to methods well known in the art. In addition, any disease agent that can be produced to have sufficient antigenic mass to effectively vaccinate nodia hatching chicks, when inactivated, would also be expected to vaccinately target egg embryos if distributed directly to the body of the embryo.

The adjuvant used in the vaccine tested in these examples was a typical commercial oil emulsion. Inactivated non-oil emulsion vaccines with adjuvants other than oil would be expected to produce an active immune response if delivered directly to the embryo body prior to hatching. Suitable adjuvants would include, but are not limited to mineral gels, polyanions, pluronic polyols, saponin derivatives, lysolecithin and other similar surfactants, glycosides and all types of oils and combinations thereof.

EXAMPLE V.

Chickens without specific pathogens (SPF) were vaccinated in egg as follows: Group 1: Phosphate buffered saline (PBS); Groups 2 and 3: 0.3 χ 109 EID50 / dose of inactivated NDV in PBS; Group 4: 0.3 χ 109 EID50 / dose of inactivated NDV mixed with an alum depositant (Imject, Pierce; aluminum hydroxide and magnesium); Group 5: A commercial NDV oil emulsion vaccine. At day 11, Group 3 patients received a second dose of NDV in PBS by subcutaneous injection. Egg-given vaccines were targeted to the embryo's body and an injection site analysis using dye was conducted on a separate group of eosimilars to estimate the percentage of embryos injected directly into the embryo's body. Egg vaccination was performed on day 19 of incubation with a 3.175 cm (1.25 inch) 23 scale needle. Fourteen per group were placed in cages and developed up to 21 days of age.

Serum samples were collected at day 21 of age and tested for NDV IgG antibody by ELISA (Idexx, Inc.). Serum samples from Groups 2, 4 and 5 were also tested for NDV-specific antibody by inhibiting hemagglutination (HI) using four HA units. The number of samples tested by Hl differed from those tested by ELISA because with several samples there was not enough serum collected to conduct the Hl test. Birds were considered to have shown a measurable antibody response (i.e. seroconverted) to vaccination if the serum sample had an ELISA titer> 200 or an Hl titer of> 3.0 Ig2 (i.e. titer 1: 8) ..

Results

Injection site analysis indicates that injections into embryo bodies accounted for 78% of all injections (Table 10).

The hatching percentage and the seroconversion rate for the NDV1 as measured by ELISA are shown in Table 11. Table 12 describes the number of birds that seroconverted using the Hl test.

From these studies, the following key points were observed. 1) NDV antigen in PBS did not stimulate an NDV ELISA measurable antibody response, even when inactivated NDV antigen was given twice as in Group 3 (once in egg again at day 11); 2) NDV-Alum (Group 4) stimulated sorption in 8/14 birds as measured by ELISA, while oil emulsion vaccination (Group 5) stimulated seroconversion in 10/13 birds when measured by ELISA; 3) NDV-Alum (Group 4) stimulated seroconversion in 12/14 when measured by Hl, whereas oil emulsion vaccine stimulated seroconversion in 11/11 of birds when measured by Hl; and 5) The Newcastle disease commercial oil emulsion vaccine (Group 5) stimulated a stronger antibody response than did the NDV-Alum vaccine (Group 4). Example III shows that egg delivery of an antigen presented in an oil emulsion adjuvant adjuvant required that the vaccine be delivered to the embryo body to stimulate an ELISA measurable antibody response. In the present example, analysis of the egg injection site indicated that 78% of eggs received the vaccine directly in the body of the embryo. EXAMPLE VI.

A study was conducted using SPF guinea fowl to determine whether the alum deposition adjuvant would stimulate an immune response when administered in egg. The groups tested were as follows: Group 1: phosphate-buffered saline (PBS) in egg, Groups 2 and 3: 1.2 χ 109 E-50 of D-propiolactone-inactivated NDV / dose in PBS in egg; Group 4: Egg administration of 1.2 χ 109 EID5O of G-propiolactone -activated NDV / dose mixed with alum at a 30% alum-to-NDV ratio: 70%. The alum used was a commercial solution of aluminum hydroxide and magnesium hydroxide (Imject, Peirce). Group 3 received an additional dose of NDV antigen in PBS at day 11 by subcutaneous injection. Administration of the vaccine to the egg was made on day 19 of incubation using a 2375 centimeter (1.25 inch) 23-scale needle. Vaccines were targeted in egg to embryo body and an injection site analysis was conducted in a separate group of similar eggs to estimate the percentage of embryos injected directly into the body of the embryo. Serum samples were removed at day 21 and tested for NDV antibody by ELISA (Idexx, Inc.). Birds were considered to have shown a measurable antibody response (i.e. seroconverted) if ELISA had a titer> 200.

Injection site analysis indicates that 81% of embryos were injected directly into the body of the embryo (Table 13). The percentage of hatching and the rate and magnitude of seroconversion against Newcastle disease virus are shown in Table 14.

These studies provided the following key points: 1) Embryos were injected into E19 and injection site data indicated that 81% of embryos were injected into the body of the embryo; and 2) In this study, alum was mixed with β-propiolactone inactivated NDV in a ratio of 30% to 70% and this differed from the study described here in Example V, in which heat-inactivated NDV was mixed at a ratio of 50%. alum to 50% NDV antigen. The difference in immune responses in the two studies may be due to differences in the NDV antigen used and / or the difference in antigen-to-alum ratios, as well as differences in ELISA used to measure antibodies.

In Example III, it was shown that the antigen presented in the depot adjuvant oil emulsion requires that the vaccine be distributed to the egg embryo body to stimulate an ELISA anti-measurable response. In this study, injection site analysis indicated that 81% of embryos were injected into the body of the embryo, and from these data it would be expected that approximately 11 out of 14 egg-vaccinated eggs would respond with an antibody response. The actual number that answered was nine out of 14.

Example VII.

A commercial oil emulsion Newcastle disease vaccine was given via the egg route to the chicks. This study determined the ability of chicks to respond to inactivated Newcastle disease virus antigens when distributed in eggs via the amniotic fluid and intra-embryo routes. Birds were bled at 13.21, 26 and 35 days of age and the NDV antibody titer was determined using ELISA (Idexx, Inc.). Injection site analysis was conducted on E18 and E19 using dye.

Hatching data are shown in Table 16. Hatching rate was normal when the oil emulsion vaccine was distributed to the body of the embryo.

The injection site (Table 15) was very accurate, with more than 90% of the embryos injected via the indicated treatment route (Table 16).

The response data for Newcastle-specific virus-specific antibody are shown in Table 17. It can be seen that asaves responded to Newcastle antigen when the vaccine was delivered to the embryo or subcutaneous egg at hatching. When NDV avacin was delivered in egg to amniotic fluid, there was no antibody response, indicating that the vaccine must be given to the embryo body to stimulate an appropriate immune response.

The foregoing is illustrative of the present invention, and is not to be construed as limiting it. The invention is defined by the following claims, with the equivalents of the claims being included therein.

Table 1. Hatching Percentage and Injection Location

<table> table see original document page 64 </column> </row> <table>

Eggs receiving Vision CD or Siteguard G demonstrated slightly lower overall hatching rates than eggs receiving control material (82.5% and 75.0% vs. 95.0%, respectively.) Table 2 Results of serum toxin neutralization:

<table> table see original document page 65 </column> </row> <table> continued

<table> table see original document page 66 </column> </row> <table>

All sera were pooled (~ 5 birds / group)

• A specific positive response was detected in egg-vaccinated birds (targeted embryo body) with the C. perfringens bacterin toxoid vaccine (Vision CD) followed by a booster after hatching. Results suggested a low antibody response (protection in birds vaccinated in egg alone (without booster after hatching).

• No protection was observed in the sera of birds vaccinated with a C. perfringens toxoid vaccine (Siteguard). Table 3. Study Summary

<table> table see original document page 67 </column> </row> <table>

Table 4. Hatching Percentage and Injection Location

<table> table see original document page 67 </column> </row> <table>

The hatching percentage of 96% was obtained after vaccination in egg with recombinant alpha toxin. An embryo bleaching of 72.16% was obtained using the 20g, 3.81 cm (1.5 ") needle. Table 5. Detection of specific antibody response via westernblot.

<table> table see original document page 68 </column> </row> <table>

• Positive and negative controls acted as expected, demonstrating the validity of the test.

• A specific antibody response was detected in the egg-vaccinated poultry (target embryo body) with the recombinant toxin-alpha formulation, with and without a booster after hatching.Table 6. Chicken antibody response to the Newcastle disease virus following administration to egg directed to the site of an oil emulsified inactivated vaccine for Newcastle disease

<table> table see original document page 69 </column> </row> <table>

Table 7: Percentage of hatchability following on-site administration of an inactivated oil emulsion vaccine for Newcastle disease

<table> table see original document page 69 </column> </row> <table> Table 8: Antibody response of chickens to Newcastle disease virus following intra-embryo egg or subcutaneous administration on hatching day of an inactivated vaccine in emulsion of

<table> table see original document page 70 </column> </row> <table>

Table 9: Percentage of hatching following intra-embryo egg administration of an inactivated oil emulsion vaccine for Newcastle disease

<table> table see original document page 70 </column> </row> <table> Table 10: Injection Location Using Dye

<table> table see original document page 71 </column> </row> <table>

Table 11: Percentage of hatching and ELISA results for NDV from birds vaccinated in egg with NDV antigen, NDV-alum antigen or an oil emulsion NDV vaccine

<table> table see original document page 71 </column> </row> <table>

** NDV-OE = Commercial Oil Emulsion Vaccine for NDV (LAHI) n / a = not applicable

Table 12: Results of NDV hemagglutination inhibition of eggs vaccinated in egg with NDV-alum antigen or an NDV vaccination in oil emulsion

<table> table see original document page 71 </column> </row> <table>

** NDV-OE = Commercial Oil Emulsion Vaccine for NDV (LAHI) Table 13: Injection Site Results

<table> table see original document page 72 </column> </row> <table>

Table 14: Percentage of hatching and ELISA results of egg chicks vaccinated with NDV or NDV-Alumen antigen,

<table> table see original document page 72 </column> </row> <table>

* Both NDV treatments were from the same group of birds that came out of the eggs; n / a = not applicable

Table 15: Injection site for site-directed egg delivery of a Newcastle disease virus oil emulsion inactivated vaccine.

<table> table see original document page 72 </column> </row> <table> Table 16: Treatment groups and hatching percentage for groups of chicks that received an oil-in-egg emulsion NDV vaccine.

<table> table see original document page 73 </column> </row> <table>

1 NDV oil emulsion - formulated for day-old chicks, 1 serving 0.1 ml

Table 17: NDV antibody response in chicks immunized with an egg oil emulsion NDV vaccine.

<table> table see original document page 73 </column> </row> <table>

1 NDV oil emulsion - formulated for day-old chicks, 1 serving in 0.1 ml <110> Doelling, Vivian W. Poston, Rebecca Heggan-Peay, Cherilyn I. Avakian, Alan P. Tyczkowski, Julius

<120> METHODS AND COMPOSITIONS FOR AVESDOMESTIC VACCINATION

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55

60

tgg gat gga aag att gat gga aca gga act cat gct atg att gta act48

Trp Asp Gly Lys Ile Asp Gly Thr Gly Thr His Wing Met Ile Val Thr15 10 15

caa ggg gtt tca till tta gaa aat gat ctg tcc aaa aat gaa cca gaa96

Gln Gly Val Ser Ile Leu Glu Asn Asp Leu Ser Lys Asn Glu Pro Glu20 25 30

agt gta aga aaa aac tta gag att tta aaa gag aac atg cat gag ctt

144

Ser Val Arg Lys Asn Leu Glu Ile Leu Lys Glu Asn Met His Glu Leu35 40 45

caa tta ggt tet act tat cca gat tat gat aag aat gea tat gat cta192

Gln Read Gly Ser Thx Tyir Piro Asp Tyir Asp Lys Asn Wing Tyir Asp Leu50 55 60

tat caa gat cat ttc tgg gat cct gat aca gat aat aat ttc tca aag240

Tyr Gln Asp His Phe Trp Asp Pro Asp Thr Asp Asn Asn Phe Ser Lys65 70 75 80

gat aat agt tgg tat tta gct tat tet ata cct gac aca ggg gaa tca288

Asp Asn Ser Trp Tyr Leu Wing Tyr Ser Ile Pro Asp Thr Gly Glu Ser85 90 95

caa ata aga aaa ttt tca gea tta gct aga tat gaa tgg caa aga gga336

Gln Ile Arg Lys Phe Be Wing Read Wing Wing Arg Tyr Glu Trp Gln Arg Gly100 105 110

aac tat aaa caa gct aca ttc tat ct gga gag gct atg cac tat ttt384

Asn Tyr Lys Gln Wing Thr Phe Tyr Leu Gly Glu Wing Met His Tyr Phe

115 120 125

gga gat ata gat act cca tat cat cct gct aat gtt act gcc gtt gat432

Gly Asp Ile Asp Pro Thr Tyr His Pro Asn Wing Val Thr Wing Asp130 135 140

age gea gga cat gtt aag tt gaa act tt gea gag gaa aga aaa gaa480

Be Wing Gly His Val Lys Phe Glu Thr Phe Wing Glu Glu Arg Lys Glu

145 150 155 160

cag tat aaa ata aac aca gea ggt tgc aaa act aat gag gat ttt tat528

Gln Tyr Lys Ile Asn Thr Wing Gly Cys Lys Thr Asn Glu Asp Phe Tyr

165 170 175

gct gat till tta aaa aac aag gat ttt aat gea tgg tca aaa gaa tat576

Wing Asp Ile Leu Lys Asn Lys Asp Phe Asn Wing Trp Ser Lys Glu Tyr

180 185 190gca aga ggt ttt gct aaa aca gga aaa tca ata tac tat agt cat gct624

Wing Arg Gly Phe Wing Lys Thr Gly Lys Ser Ile Tyr Tyr Ser His Ala195 200 205

age atg agt cat agt tgg gat gat tgg gat tat gca gca aag gta act672

Be Met Be His Ser Trp Asp Asp Trp Asp Tyr Wing Wing Lys Val Thr210 215 220

tta gct aac tet caa aaa gga aca gca gga tat att tat aga ttc tta720

Leu Wing Asn Be Gln Lys Gly Thr Wing Gly Tyr Ile Tyr Arg Phe Leu

225 230 235 240

cac gat gta tca gag ggt aat gat cca tca gt gga aag aat gta aaa768

His Asp Val Be Glu Gly Asn Asp Pro Be Val Gly Lys Asn Val Lys245 250 255

gaa cta gta gct tac ata tca act agt ggt gaa aaa gat gct gga aca816

Glu Leu Val Wing Tyr Ile Ser Thr Be Gly Glu Lys Asp Wing Gly Thr

260 265 270

gat gac tac atg tat ttt834

Asp Asp Tyr Met Tyr Phe275

<210> 6

<211> 278

<212> PRT

<213> Clostridium perfringens

<400> 6

Trp Asp Gly Lys Ile Asp Gly Thr Gly Thr

1 5 10 15

Gln Gly Val Ser Ile Leu Glu Asn Asp Leu Ser Lys Asn Glu Pro Glu20 25 30

Ser Val Arg Lys Asn Leu Glu Ile Leu Lys Glu Asn Met His Glu Leu35 40 45

Gln Leu Gly Be Thr Tyr Pro Asp Tyr Asp Lys Asn Wing Tyr Asp Leu50 55 60

55 Tyr Gln Asp His Phe Trp Asp Pro Asp Thr Asp Asn Asn Phe Ser Lys65 70 75 80

Asp Asn Be Trp Tyr Leu Wing Tyr Be Ile Pro Asp Thr Gly Glu Ser60 85 90 95Gln Ile Arg Lys Phe Be Wing Leu Arg Wing Tyr Glu Trp Gln Arg Gly100 105 110

Asn Tyr Lys Gln Wing Thr Phe Tyr Leu Gly Glu Wing Met His Tyr Phe115 120 125

Gly Asp Ile Asp Pro Thr Tyr His Pro Asn Wing Val Thr Wing Asp130 135 140

Ser Wing Gly His Val Lys Phe Glu Thr Phe Wing Glu Glu Arg Lys Glu145 150 155 160

Gln Tyr Lys Ile Asn Thr Wing Gly Cys Lys Thr Asn Glu Asp Phe Tyr165 170 175

Asp Wing Ile Read Lys Asn Lys Asp Phe Asn Trp Wing Be Lys Glu Tyr180 185 190

Wing Arg Gly Phe Wing Lys Thr Gly Lys Ser Ile Tyr Tyr Ser His Ala195 200 205

Be Met Be His Ser Trp Asp Asp Trp Asp Tyr Wing Wing Lys Val Thr210 215 220

Leu Wing Asn Be Gln Lys Gly Thr Wing Gly Tyr Ile Tyr Arg Phe Leu225 230 235 240

His Asp Val Be Glu Gly Asn Asp Pro Be Val Gly Lys Asn Val Lys245 250 255

Glu Leu Val Wing Tyr Ile Ser Thr Be Gly Glu Lys Asp Wing Gly Thr260 265 270

Asp Asp Tyr Met Tyr Phe275

<210> 7

<211> 120

<212> DNA

<213> Clostridium perfringens

<220> <221> CDS <222> (1) .. (120) <400> 7

tac ata tca act agt ggt gaa aaa gat gct gga aca gat gac tac atg48

Tyr Ile Be Thr Be Gly Glu Lys Asp Wing Gly Thr Asp Asp Tyr Met15 10 15

tat ttt gga till aaa aca aag gat gga aaa act caa gaa tgg gaa atg96

Tyr Phe Gly Ile Lys Thr Lys Asp Gly Lys Thr Gln Glu Trp Glu Met20 25 30

gac aac cca gga aat gat ttt atg120

Asp Asn Pro Gly Asn Asp Phe Met35 40

<210> 8 <211> 40 <212> PRT

<213> Clostridium perfringens <400> 8

Tyr Ile Be Thr Be Gly Glu Lys Asp Wing Gly Thr Asp Asp Tyr Met15 10 15

Tyr Phe Gly Ile Lys Thr Lys Asp Gly Lys Thr Gln Glu Trp Glu Met20 25 30

Asp Asn Pro Gly Asn Asp Phe Met35 40

<210> 9

<211> 1197

<212> DNA

<213> Clostridium perfringens

<220>

<221> CDS <222> (1) .. (1194)

<400> 9

atg aaa aga aag att tgt aag gcg ctt att tgt gct acg cta gea act48

Met Lys Arg Lys Ile Cys Lys Wing Read Ile Cys Wing Thr Read Wing Thr15 10 15

age cta tgg gct ggg gea tca act aaa gtc tac gct tgg gat gga aag96

Ser Leu Trp Wing Gly Wing Ser Thr Lys Val Tyr Wing Trp Asp Gly Lys20 25 30

att gat gga aca gga act cat gct attg att gta act caa ggg gtt tca144

Ile Asp Gly Thr Gly Thr His Wing Met Ile Val Thr Gln Gly Val Ser35 40 45

until tta gaa aat gat ctg tcc aaa aat gaa cca gaa agt gta aga aaa192

Ile Leu Glu Asn Asp Leu Be Lys Asn Glu Pro Glu Be Val Arg Lys50 55 60

aac tta gag att tta aaa gag aac atg cat gag ctt caa tta ggt tet240

Asn Leu Glu Ile Leu Lys Glu Asn Met His Glu Leu Gln Leu Gly Ser65 70 75 80

act cca tat gat gat gat aag aat gea tat gat cta tat caa gat cat288

Thr Tyr Pro Asp Tyr Asp Lys Asn Wing Tyr Asp Read Tyr Gln Asp His85 90 95

ttc tgg gat cct gat aa gat aat aat ttc tca aag gat aat agt tgg336

Phe Trp Asp Pro Asp Thr Asp Asn Ashe Phe Ser Lys Asp Asn As Trp100 105 110

tat tta gct tat tet ata cct gac aca ggg gaa tca caa ata aga aaa384

Tyr Leu Wing Tyr Ser Ile Pro Asp Thr Gly Glu Ser Gln Ile Arg Lys

115 120 125

ttt tca gea tta gct aga tat gaa tgg caa aga gga aac tat aaa caa432

Phe Ser Wing Read Wing Arg Tyr Glu Trp Gln Arg Gly Asn Tyr Lys Gln130 135 140

gct aca ttc tat ct gga gag gct atg cac tat ttt gga gat ata gat480

Wing Thr Phe Tyr Leu Gly Glu Wing Met His Tyr Phe Gly Asp Ile Asp

145 150 155 160

act cca tat cat cct gct aat gtt

Thr Pro Tyr His Wing Asn Val Wing Thr Wing Val Asp Be Wing Gly His

165 170 175

gtt aag ttt gaa act ttt gea gag gaa aga aaa gaa cag tat aaa ata576

Val Lys Phe Glu Thr Phe Wing Glu Glu Arg Lys Glu Gln Tyr Lys Ile180 185 190

aac aca gea ggt tgc aaa act aat gag gat ttt tat gct gat by tta624

Asn Thr Wing Gly Cys Lys Thr Asn Glu Asp Phe Tyr Wing Asp Ile Leu

195 200 205

aaa aac aag gat ttt aa gea tgg tca aaa gaa tat gea aga ggt ttt672

Lys Asn Lys Asp Phe Asn Wing Trp Being Lys Glu Tyr Wing Arg Gly Phe210 215 220

gct aaa aca gga aaa tca ata tac tat agt cat gct age atg agt cat720

Wing Lys Thr Gly Lys Be Ile Tyr Tyr Be His Wing Be Met Be His225 230 235 240

agt tgg gat gat tgg gat tat gca gca aag gta act tta gct aac tct768

Ser Trp Asp Asp Trp Asp Tyr Wing Wing Lys Val Thr Read Wing Wing Asn

245 250 255

caa aaa gga aca gca gga tat att tat aga ttc tta cac gat gta tca816

Gln Lys Gly Thr Wing Gly Tyr Ile Tyr Arg Phe Read His Asp Val Ser260 265 270

gag ggt aat gat cca tca gtt gga aag aat gta aaa gaa cta gta gct864

Glu Gly Asn Asp Pro To Be Val Gly Lys Asn Val Lys Glu Leu Val Ala275 280 285

tac ata tca act agt ggt gaa aaa gat gct gga aca gat gac tac atg912

Tyr Ile Be Thr Be Gly Glu Lys Asp Wing Gly Thr Asp Asp Tyr Met290 295 300

tat ttt gga till aaa aca aag gat gga aaa act caa gaa tgg gaa atg960

Tyr Phe Gly Ile Lys Thr Lys Asp Gly Lys Thr Gln Glu Trp Glu Met

305 310 315 320

gac aac cca gga aat gat ttt atg act gga agt aaa gac act tat act1008

Asp Asn Pro Gly Asn Asp Phe Met Thr Gly Ser Lys Asp Thr Tyr Thr

325 330 335

ttc aaa tta aaa gat gaa aat cta aaa att gat gat aa caa aat atg1056

Phe Lys Leu Lys Asp Glu Asn Leu Lys Ip Asp Ile Gln Asn Met

340 345 350

tgg att aga aaa aga aaa tat aca gca ttc cca gat gct tat aag cca1104

Trp Ile Arg Lys Arg Lys Tyr Thr Wing Phe Pro Asp Wing Tyr Lys Pro355 360 365

gaa aac ata aag ata ata gca aat gga aaa gtt gta gta gac aaa gat1152

Glu Asn Ile Ile Ile Ile Wing Asn Gly Lys Val Val Val Val Asp Lys Asp370 375 380

aat gag tgg att tca gga aat tca act tat aat aaa taa1197

Ile Asn Glu Trp Ile Be Gly Asn Be Thr Tyr Asn Ile Lys385 390 395

<210> 10 <211> 398

<212> PRT

<213> Clostridium perfringens

<400> 10Met Lys Arg Lys Ile Cys Lys Wing Leu Ile Cys Wing Thr Leu Wing Thr15 10 15

Ser Leu Trp Wing Gly Wing Ser Thr Lys Val Tyr Wing Trp Asp Gly Lys20 25 30

Ile Asp Gly Thr Gly Thr His Wing Met Ile Val Thr Gln Gly Val Ser35 40 45

Ile Leu Glu Asn Asp Leu Be Lys Asn Glu Pro Glu Be Val Arg Lys50 55 60

Asn Leu Glu Ile Leu Lys Glu Asn Met His Glu Leu Gln Leu Gly Ser65 70 75 80

Thr Tyr Pro Asp Tyr Asp Lys Asn Wing Tyr Asp Read Tyr Gln Asp His85 90 95

Phe Trp Asp Pro Asp Thr Asp Asn Ashe Phe Ser Lys Asp Asn As Trp100 105 110

Tyr Leu Wing Tyr Ser Ile Pro Asp Thr Gly Glu Ser Gln Ile Arg Lys115 120 125

Phe Ser Wing Read Wing Arg Tyr Glu Trp Gln Arg Gly Asn Tyr Lys Gln130 135 140

Thr Phe Wing Tyr Leu Gly Glu Met Wing His Tyr Phe Gly Asp Ile Asp145 150 155 160

Thr Pro Tyr His Pro Asn Val Wing Thr Val Asp Ser Wing Gly His165 170 175

Val Lys Phe Glu Thr Phe Wing Glu Glu Arg Lys Glu Gln Tyr Lys Ile180 185 190

Asn Thr Wing Gly Cys Lys Thr Asn Glu Asp Phe Tyr Wing Asp Ile Leu195 200 205

Lys Asn Lys Asp Phe Asn Wing Trp Being Lys Glu Tyr Wing Arg Gly Phe210 215 220

Lys Thr Wing Gly Lys Be Ile Tyr Tyr Be His Wing Be Met Be His225 230 235 240Ser Trp Asp Asp Trp Asp Tyr Wing Lys Val Thr Wing Leu Wing Asn Ser245 250 255

Gln Lys Gly Thr Wing Gly Tyr Ile Tyr Arg Phe Read His Asp Val Ser260 265 270

Glu Gly Asn Asp Pro To Be Val Gly Lys Asn Val Lys Glu Leu Val Ala10 275 280 285

Tyr Ile Be Thr Be Gly Glu Lys Asp Wing Gly Thr Asp Asp Tyr Met290 295 300

Tyr Phe Gly Ile Lys Thr Lys Asp Gly Lys Thr Gln Glu Trp Glu Met305 310 315 320

Asp Asn Pro Gly Asn Asp Phe Met Thr Gly Ser Lys Asp Thr Tyr Thr325 330 335

Phe Lys Leu Lys Asp Glu Asn Leu Lys Ile Asp Asp Ile Gln Asn Met340 345 350

Trp Ile Arg Lys Arg Lys Tyr Thr Wing Phe Pro Asp Wing Tyr Lys Pro

30 355 360 365

Glu Asn Ile Ile Ile Ile Wing Asn Gly Lys Val Val Val Val Asp Lys Asp370 375 380

Ile Asn Glu Trp Ile Be Gly Asn Be Thr Tyr Asn Ile Lys385 390 395

<210> 11 <211> 336 <212> PRT

<213> Clostridium perfringens <400> 11

Met Lys Lys Lys Phe Ile To Be Leu Val Ile Val To Be Leu Leu Asn15 10 15

Gly Cys Leu Read Be Pro Arg Leu Val Tyr Wing Asn Asp Ile Gly Lys20 25 30

Thr Thr Thr Ile Thr Arg Asn Lys Thr Be Asp Gly Tyr Thr Ile Ile35 40 45

Thr Gln Asn Asp Lys Trp Ile Ile Ser Tyr Gln Ser Val Asp Ser Ser 55 55 60

Ser Lys Asn Glu Asp Gly Phe Thr Wing Ser Ile Asp Wing Arg Phe Ile65 70 75 80

Asp Asp Lys Tyr Be Being Glu Met Thr Thr Read Ile Asn Read Thr Thr Gly85 90 95

Phe Met Being Ser Lys Lys Glu Asp Val Ile Lys Lys Tyr Asn Leu His100 105 110

Asp Asn Thr Asn Ser Thr Ile Wing Asn Phe Pro Val Arg Tyr Ser Ile115 120 125

Ser Ile Leu Asn Glu Ser Ile Asn Glu Asn Val Lys Ile Val Asp Ser130 135 140

Ile Pro Lys Asn Thr Ile Be Gln Lys Thr Val Be Asn Thr Met Gly145 150 155 160

Tyr Lys Ile Gly Gly Be Ile Glu Ile Glu Glu Asn Lys Pro Lys Ala165 170 175

Ser Ile Glu Ser Glu Tyr Wing Glu Ser Ser Thr Ile Glu Tyr Val Gln180 185 190

Pro Asp Phe Ser Thr Ile Gln Thr Asp His Ser Thr Be Lys Wing Ser195 200 205

Trp Asp Thr Lys Phe Thr Glu Thr Thr Gly Asn Tyr Asn Leu Lys210 215 220

Ser Asn Asn Pro Val Tyr Gly Asn Glu Met Phe Met Tyr Gly Arg Tyr225 230 235 240

Thr Asn Val Pro Wing Thr Glu Asn Ile Ile Pro Asp Tyr Gln Met Ser245 250 255

Lys Leu Ile Thr Gly Gly Leu Asn Pro Asn Met Ser Val Val Leu Thr260 265 270

Pro Wing Asn Gly Thr Glu Glu Ser Ile Ile Lys Val Lys Met Glu Arg275 280 285

Glu Arg Asn Cys Tyr Tyr Read Asn Trp Asn Gly Wing Asn Trp Val Gly290 295 300

Gln Val Tyr Be Arg Leu Wing Phe Asp Thr Pro Asn Val Asp Be His 305 310 315 320

Ile Phe Thr Phe Lys Ile Asn Trp Read Thr His Lys Val Thr Wing Ile325 330 335

<210> 12 <211> 328 <212> PRT <213> Clostridium perfringens <400> 12

Met Lys Lys Asn Leu Val Lys Ser Leu Wing Ile Wing Ser Wing Val Ile 1 5 10 15

Ser Ile Tyr Ser Ile Val Asn Ile Val Ser Pro Thr Asn Val Ile Wing 25 25

Lys Glu Ile Be Asn Thr Val Be Asn Glu Met Be Lys Lys Wing Ser35 40 45

Tyr Asp Asn Val Asp Thr Read Ile Glu Lys Gly Arg Tyr Asn Thr Lys50 55 60

35 Tyr Asn Tyr Read Lys Arg Met Glu Lys Tyr Tyr Pro Asn Ala Met Ala65 70 75 80

Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly Asn Asp Phe Tyr Ile 85 90 95

Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro Ser Met Asn Tyr Leu100 105 110

Glu Asp Val Tyr Val Gly Lys Wing Read Leu Thr Asn Asp Thr Gln Gln115 120 125

Glu Gln Lys Read Lys Be Gln Be Phe Thr Cys Lys Asn Thr Asp Thr130 135 140

55 Val Thr Wing Thr Thr Thr His Thr Val Gly Thr Ser Ile Gln Wing Thr145 150 155 160

Lys Wing Phe Thr Val Pro Phe Asn Glu Thr Gly Val Ser Leu Thr Thr60 165 170 175Ser Tyr Ser Phe Wing Asn Thr Asn Thr Asn Thr Asn Ser Lys Glu Ile180 185 190

Thr His Asn Val Pro Be Gln Asp Ile Leu Val Pro Wing Asn Thr Thr195 200 205

Val Glu Val Ile Wing Tyr Leu Lys Lys Val Asn Val Lys Gly Asn Val210 215 220

Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp Gly Glu Ile Pro Ser225 230 235 240

Tyr Leu Wing Phe Pro Arg Asp Gly Tyr Lys Phe Be Read Asp Thr245 250 255

Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr Ile Asn Ile Asn Gly260 265 270

Lys Gly Asn Tyr Ser Val Wing Gly Asp Glu Leu Ile Val Lys Val275 280 285

Arg Asn Read Asn Thr Asn Asn Val Gln Glu Tyr Val Ile Pro Val Asp290 295 300

Lys Lys Glu Lys Be Asn Asp Be Asn Ile Val Lys Tyr Arg Be Leu305 310 315 320

Tyr Ile Lys Pro Wing Gly Ile Lys325

<210> 13 <211> 283 <212> PRT

<213> Clostridium perfringens <400> 13

Lys Ala Ser Tyr Asp Asn Val Asp Thr Read Ile Glu Lys Gly Arg Tyr15 10 15

Asn Thr Lys Tyr Asn Tyr Read Lys Arg Met Met Glu Lys Tyr Tyr Pro Asn20 25 30

Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly Asn Asp35 40 45Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro Ser Met50 55 60

Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Wing Leu Leu Thr Asn Asp65 70 75 80

Thr Gln Gln Glu Gln Lys Read Lys Be Gln Be Phe Thr Cys Lys Asn

85 90 95

Thr Asp Thr Val Thr Wing Thr Thr His Thr Val Gly Thr Ser Ile

100 105 110

Gln Wing Thr Wing Wing Lys Phe Thr Val Phe Asn Glu Thr Gly Val Ser115 120 125

Read Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr Asn Ser130 135 140

Lys Glu Ile Thr His Asn Val Pro Be Gln Asp Ile Leu Val Pro Ala145 150 155 160

Asn Thr Thr Val Glu Val Ile Wing Tyr Leu Lys Lys Val Asn Val Lys

165 170 175

Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp Gly Glu

180 185 190

Ile Pro Ser Tyr Leu Wing Phe Pro Arg Asp Gly Tyr Lys Phe Ser Leu195 200 205

Be Asp Thr Val Asn Lys Be Asp Leu Asn Glu Asp Gly Thr Ile Asn210 215 220

Ile Asn Gly Lys Gly Asn Tyr Ser Wing Val Met Gly Asp Glu Leu Ile225 230 235 240

Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln

245 250 255

Pro Val Asp Lys Lys Glu Lys Being Asn Asp Being Asn Ile

260 265 270

Arg Ser Leu Tyr Ile Lys Pro Wing Gly Ile Lys275 280

Claims (70)

A method of immunizing an avian patient against a pathogen comprising administering to egg during the final fourth incubation an effective immunizing dose of a composition which induces an immune response against the pathogen, wherein the immunogenic composition is administered by egg injection directly into the egg. embryo. The method of claim 1, wherein the composition is administered directly to skeletal muscle tissue in the embryo. The method of claim 2, wherein the skeletal muscle tissue is selected from the group consisting of tiny chest tissue and mature egg muscle tissue. The method of claim 1, wherein the composition is administered directly to the embryo on the head, neck, shoulder, wing, back, chest, leg or any combination thereof. The method of claim 1, wherein the composition is administered to the embryo subcutaneously, intradermally, intravenously, intramuscularly, intra-abdominally or any combination thereof. The method of claim 5, wherein the composition is administered to the embryo subcutaneously. The method of claim 5, wherein the composition is administered to the embryo intra-abdominally. The method of claim 1, wherein the avian patient is a small picture. The method of claim 8, wherein the composition is administered from day 15 to day 20 of incubation. The method of claim 8, wherein the composition is administered on day 18 or 19 of incubation. The method of claim 1, wherein the composition comprises a water in oil in water emulsion. The method of claim 1, wherein the composition is administered in a needle having a length of 1.905 centimeters to -10.16 centimeters (¾ inches to 4 inches). The method of claim 12, wherein the needle has a scaling from 15 to 28. The method of claim 12, wherein the needle has a non-pointed end. The method of claim 12, wherein the needle has a sharp end. The method of claim 15, wherein the needle has an inclination at an angle ranging from about 10 ° to about 45 °. The method of claim 12, wherein the needle passes through the shell at the large end of an egg at an angle deviation from about 1 ° to about 20 ° from the long axis of the egg. The method of claim 1, further comprising the step of administering a booster dose of the composition to the patient after hatching. The method of claim 1, wherein the composition comprises an adjuvant. The method of claim 19, wherein the adjuvant comprises an aluminum derived adjuvant, a saponin, mineral gels, polyanions, pluronic polyols, saponin derivatives, lysolecithin and other similar surfactants, glycosides, all types of oils and any combination thereof. The method of claim 1, wherein the composition induces an immune response to treat and / or prevent coccidiosis, Marek's disease, infectious bursal disease, Newcastle disease, avian smallpox infection, Clostridium spp. Infection, avian influenza, infectious bronchitis, chick anemia virus infection, laryngotracheitis, avian pneumovirus infection, avian reovirus infection, avian adenovirus infection, rotavirus infection, astrovirus infection, inclusion body hepatitis, posture syndrome, Escherichia coli infection, Mucoplasma infection spp., Salmonella infection spp., Campylobacter spp. infection, Haemophillus spp. infection, Listeriaspp infection., Pasteurella spp. and any combination of these. The method of claim 1, wherein the composition comprises a non-replicating agent that induces an immune response against the pathogen. The method of claim 1, wherein an immunizing-effective dose of two or more compositions that induce an immune response against the pathogen is administered in egg to the embryo, wherein the two or more compositions are administered simultaneously or sequentially in any order. The method of claim 1, wherein an effective immunizing dose of two or more compositions that elicit a pathogen immune response is administered in egg and at least one composition is administered to the embryo, wherein the two or more compositions are administered simultaneously or sequentially in either order. The method of claim 24, wherein at least one composition is administered to ammonium, amniotic fluid, embryo body, yolk sac or any combination thereof. The method of claim 1, further comprising administering to an egg an immune stimulant at any time during incubation, wherein the immune stimulant and the composition are administered simultaneously or sequentially in any order. The method of claim 1, further comprising administering to egg a nutrient formulation, an enteric modulator, or a combination thereof at any time during incubation, wherein the nutrient formulation, the enteric modulator or a combination thereof and administration are administered simultaneously or sequentially at any time. order. The method of claim 1, wherein the composition is administered in egg with an automation injection device. 29. A method of immunizing an avian patient against Clostridium infection, comprising administering to egg, during the fourth final incubation, an effective immunizing dose of an immunogenic composition that induces an immune response against a species of Clostridium, where the immunogenic composition is administered by injection into The method of claim 29, wherein the immunogenic composition is administered to ammonium. The method of claim 30, wherein the immunogenic composition is administered axially through the large end of the amniotic fluid. The method of claim 29, wherein the immunogenic composition is administered directly to the body of the embryo. The method of claim 32, wherein the immunogenic composition is administered to the embryo parenterally. The method of claim 29, wherein the avian patient is a small picture. The method of claim 34, wherein the immunogenic composition is administered from day 15 to day 20 of incubation. The method of claim 34, wherein the immunogenic composition is administered on day 18 or 19 of incubation. The method of claim 29, wherein the avian patient is an expert. The method of claim 29, wherein the immunogenic composition comprises a Clostridium perfringens toxoid. The method of claim 29, wherein the immunogenic composition comprises a Clostridium perfringens bacterin. The method of claim 29, wherein the immunogenic composition comprises a Clostridium perfringens toxoid and a Clostridium perfringens bacterine. The method of claim 29, wherein the immunogenic composition comprises a Clostridium perfringens toxin. The method of claim 41, wherein the Clostridiumperfringens toxin is a Clostridium perfringens alpha toxin. The method of claim 29, wherein the immunogenic composition comprises an attenuated Clostridium perfringens. The method of claim 29, wherein the immunogenic composition comprises a water in oil in water emulsion. The method of claim 29, wherein the immunogenic composition comprises an adjuvant. The method of claim 45, wherein the adjuvant comprises an aluminum-derived adjuvant, a saponin, an oil, or any combination of the foregoing. The method of claim 29 further comprising administering to an egg an immune stimulant at any time during incubation. The method of claim 29, further comprising administering in egg a coccidiosis vaccine, a Marek disease vaccine, an infectious bursal disease vaccine, a Newcastle disease vaccine, an avian smallpox vaccine, or any combination of the foregoing. The method of claim 48, wherein the immunogenic composition and vaccine are administered simultaneously. The method of claim 48, wherein the immunogenic composition and vaccine are administered in the same formulation. The method of claim 29, further comprising administering to egg an nutrient formulation, an enteric modulator, or a combination thereof. 52. An immunogenic composition comprising an effective dose-immunizing of an attenuated Clostridium perfringens in a pharmaceutically acceptable carrier. The immunogenic composition of claim 52, wherein the immunogenic composition comprises a water in oil in water emulsion. The immunogenic composition of claim 52, wherein the immunogenic composition comprises an adjuvant. The immunogenic composition of claim 52, wherein the immunogenic composition further comprises a decoccidiosis vaccine, a Marek's disease vaccine, a bursal infectious disease vaccine, a Newcastle disease vaccine, a smallpox vaccine, or any combination of the foregoing. The immunogenic composition of claim 52, wherein the composition further comprises a nutrient formulation, an enteric modulator, or a combination thereof. 57. An immunogenic composition comprising in a pharmaceutically acceptable vehicle: (a) an effective immunizing dose of a Clostridium perfringens toxoid, a Clostridium perfringens bacterin, a C. perfringens toxin, or any combination of the foregoing; and (b) an effective immunizing dose of a decoccidiosis vaccine, a Marek's disease vaccine, a bursal infectious disease vaccine, a Newcastle disease vaccine, a smallpox vaccine, or any combination of the foregoing. 58. An immunogenic composition comprising in a pharmaceutically acceptable vehicle: (a) an effective immunizing dose of a Clostridium perfringens toxoid, a Clostridium perfringens bacterin, a C. perfringens toxin, or any combination of the foregoing; and (b) a water in oil in water emulsion. 59. An immunogenic composition comprising in a pharmaceutically acceptable vehicle: (a) an effective immunizing dose of a Clostridium perfringens toxoid, a Clostridium perfringens bacterin, a C. perfringens toxin, or any combination of the foregoing; and (b) an adjuvant comprising an aluminum-derived adjuvant, a saponin, an oil, or any combination of the foregoing. 60. A method of immunizing an avian patient against Clostridium infection, comprising administering to the avian patient an effective immunizing dose of a Clostridium toxin bacterin composition by egg injection during the final fourth incubation. The method of claim 60, further comprising the step of administering a booster dose of the Clostridium bacterin toxoid composition to the avian patient after hatching. The method of claim 60, wherein the avian patient is a small picture. The method of claim 60, wherein the composition further comprises an adjuvant. The method of claim 60, wherein the composition comprises a Vision CD® vaccine. 65. A method of immunizing an avian patient against Clostridium infection, comprising administering to the avian patient an effective immunizing dose of a recombinant toxin or its Clostridium immunogenic fragment by injection into an egg during the final fourth incubation. The method of claim 65, further comprising the step of administering a booster dose of the recombinant toxin or immunogenic fragment thereof to the avian patient upon hatching. The method of claim 65, wherein the avian patient is a small picture. The method of claim 65, wherein the toxin is administered as an adjuvant. The method of claim 68, wherein the adjuvant is incomplete Freund's Quil A adjuvant. 70. A method of immunizing an avian patient against enteritenecrotic, comprising administering to egg during the final fourth incubation an effective immunizing dose of an immunogenic composition which induces an immune response against a species of Clostridium, where immunogenic composition is administered by egg injection. .